Polymorphisms in the human gene for cytochrome P450 polypeptide 2C8 and their use in diagnostic applications

ABSTRACT

The present invention relates to a polymorphic CYP2C8-polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a prodrug or drug or an inhibitor are also encompassed by the present invention. In addition, the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease. Further, the invention relates to a method of detection of the polynucleotide of the invention. Furthermore, comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention. Finally, the invention relates to a diagnostic kit.

The present invention relates to a polymorphic CYP2C8 polynucleotide.Moreover, the invention relates to genes or vectors comprising thepolynucleotides of the invention and to a host cell geneticallyengineered with the polynucleotide or gene of the invention. Further,the invention relates to methods for producing molecular variantpolypeptides or fragments thereof, methods for producing cells capableof expressing a molecular variant polypeptide and to a polypeptide orfragment thereof encoded by the polynucleotide or the gene of theinvention or which is obtainable by the method or from the cellsproduced by the method of the invention. Furthermore, the inventionrelates to an antibody which binds specifically the polypeptide of theinvention. Moreover, the invention relates to a transgenic non-humananimal. The invention also relates to a solid support comprising one ora plurality of the above mentioned polynucleotides, genes, vectors,polypeptides, antibodies or host cells. Furthermore, methods ofidentifying a polymorphism, identifying and obtaining a pro-drug or drugor an inhibitor are also encompassed by the present invention. Inaddition, the invention relates to methods for producing of apharmaceutical composition and to methods of diagnosing a disease.Further, the invention relates to a method of detection of thepolynucleotide of the invention. Furthermore, comprised by the presentinvention are a diagnostic and a pharmaceutical composition. Even more,the invention relates to uses of the polynucleotides, genes, vectors,polypeptides or antibodies of the invention. Finally, the inventionrelates to a diagnostic kit.

Cytochrome P450 enzymes are metabolic enzymes differentially expressedin several tissues. Cytochrome P450 2C mRNA was detected in abundance inhepatic tissue, to a lesser extend in extrahepatic tissues, e.g. kidney,adrenal gland, brain, uterus, mammary gland, ovary and duodenum, butneither in testes nor ovary (Klose, J Biochem Mol Toxicol 13 (1999),289-95). Of the CYP2C subfamily, clustered on chromosome 10q24.1 (Gray,Genomics 28 (1995), 328-32), CYP2C9 and 2C19 are those which gainedmajor interest due to their prominent role in metabolizing therapeuticdrugs. Differential breakdown of their substrates led to theidentification of alleles for poor (PM) or extensive metabolizers (EM).Nevertheless, the existence of minor CYP2C8 genes was known andcharacterized to display about 90% amino acid homology (Goldstein,Pharmacogenetics 4 (1994), 285-99). Only recently, the genomic sequenceof CYP2C8, spanning a 31 kb region, was published. Interestingly, thegene is involved in intergenic splicing with CYP2C18 composed of 9 exons(Finta, Genomics 63 (2000), 433-8).

Arachidonic acid is one major endogenous substrate for CYP2C8 andspecifically epoxidated to equivalent forms of 11, 12- and 14,15-epoxides. Concerning xenobiotical substrates CYP2C8 represents theisoform with the narrowest substrate specificity. The anticancer drugTAXOL (paclitaxel), also well known to be a substrate for MDR-1(Mechetner, Clin Cancer Res. 4 (1998), 389-398), is known to be theprototype. Several other drugs, e.g. verapamil (Tracy, Br J ClinPharmacol. 47 (1999), 545-52) and rosiglitazone (Malinowski, Clin Ther.22 (2000), 1151-68) are preferable substrates for CYP2C8 in comparisonto other CYP2Cs or CYP3As. Drugs like benzphetamine, retinoic acid,tolbutamide, benzo(a)pyrene, carbamazepine and R-ibuprofen represent aminor contribution of CYP2C8 (Wrighton, J Clin Invest. 80 (1987),1017-22; Relling, J Pharmacol Exp Ther. 252 (1990), 442-7; Hamman,Biochem Pharmacol. 54 (1997), 33-41; Kerr, Biochem Pharmacol. 47 (1994),1969-79; Yun, Cancer Res. 52 (1992), 1868-74; Leo, Arch Biochem Biophys259 (1987), 241-9). So far, the enzymatic induction has only be observedby phenobarbital and rifampicin (Morel, Eur J. Biochem. 191 (1990),437-44). Thum and Borlak (Thum and Borlak, Br J Pharmacol 130 (2000),1745-52) found a strong correlation between tissue specific geneexpression and enzyme activity. Increased CYP2C8 mRNA expression withinthe right heart ventricle might explain for the lack of efficacy ofcardioselective drugs like verapamil. In a porcine system, Fisslthaler(Fisslthaler, Nature 401 (1999), 493-7; Fisslthaler, Semin Perinatol 24(2000), 15-9; Fisslthaler, Circ Res. 88 (2001), 44-51) could show thatCYP2C8 meets all criteria for the coronary endothelium-derivedhyperpolarisation factor synthase acting on vascular smooth muscle cellsprior to dilation.

Since the mRNA has been published, first single nucleotide polymorphisms(SNPs) in exons 3, 5 and 8 were reported in an abstract (Goldstein,Microsomes and Oxidation, Stresa (2000), Italy): an exchange in position139 of Arg to Lys (exon 3) could be linked to a SNP in exon 8(Lys399Arg), occurring primarily in Caucasians, and correlated to poormetabolizing phenotype (PM). Exon 5 displays a mutation (Iso269Phe) thatis associated with poor metabolizing enzyme restricted toAfrican-Americans. The regulation of the 2Cs is supposed to be modifiedby polymorphisms in the untranslated region. Regarding CYP2C8, twopreviously unidentified transcription regulatory factor sites for C/EBPand HPF-1, but no relevant SNPs were identified by Goldstein andcoworkers (Goldstein, Microsomes and Oxidation, Stresa Italy (2000),Italy) in that region.

However, means and methods for reliable and improved diagnosing andtreating a variety of diseases and disorders or for predicting andovercoming undesired drug effects or interactions based on dysfunctionsor dysregulations of cytochrome 2C8 variants were not available yet butare nevertheless highly desirable. Thus, the technical problemunderlying the present invention is to comply with the above specifiedneeds.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Correlation of the SNP C104G (Exon 5, 1264M) with reduced protein levelsof CYP2C8. Expression levels of 14 individuals were determined byWestern Blot analysis and LC-MS using verapamil as specific substrate.The boxplots show the distribution of samples according to the genotypeat amino acid position 264. The genotype-phenotype correlation issignificant (p=0.037, N=14).

FIG. 2

Correlation of the SNP −370 relative to the start codon ATG withincreased expression levels as detected by western blotting, using thedrug TAXOL (paclitaxel) as specific substrate. As shown in the boxplotsthe genotype-phenotype correlation is significant between homozygouswild type and heterozygous mutant samples (p=0.044, N=20). (Onehomozygous sample was analyzed and yielded expression levels >400).

FIG. 3

Correlation of the SNP at position −370 in the untranslated region basedon deviates from mean values of CYP2C8 expression levels from twoindependent sample collectives (N=62). The phenotype of the patients wasdetermined by LC/MS and Western Blot. The phenotype-genotype correlationis significant as shown in the boxplots (p=0.017 for homozygous vswildtype and p=0.071 for heterozygous vs wildtype).

FIG. 4

Correlation of the allele including the linked SNPs G-1207A,delAT-640/41 (both in the promoter), G270A (exon 3), and A206G (exon 8)with a poor metabolizer phenotype (PM). However, no homozygousindividuals for this allele could yet be phenotyped. The data shown inthe box plot do only show a trend (p=0,071; N=18) but no significance.

FIG. 5

Transfection of LS174T cells with either a CYP2C8 wild type promoterconstruct or two SNP(s) containing promoter fragments (G-1207A and −640to 641delAT or T-370G). Using the eukaryotic pGL3 expression vector meanvalues of six independent transfection assays were analysed followingnormalisation of 2C8 wild type activity to 100%.

FIG. 6

Computer modeled CYP2C8 enzyme structure of the protein variants Thr 159Pro (frameshift), Glu 274 Stop and Gly 365 Ser. Dark grey represents theunchanged structure of the variant protein, the light grey prepresentsthe missing amino acids of the CYP2C8 variant structure. The circleindicates the active site of the enzyme with the altered amino acid Gly365 Ser in dark.

FIG. 7A-D

Reference or wild type GenBank sequences for the polynucleotides,polypeptide and mRNA according to the present invention (SEQ ID NOS:400-417, respectively, in order of appearance).

Accordingly, the present invention relates to a polynucleotidecomprising a polynucleotide selected from the group consisting of:

-   (a) a polynucleotide having the nucleic acid sequence of SEQ ID NO:    54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102,    105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141,    144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180,    183, 186, 189, 192, 195, 198, 201, 210, 213, 216, 219, 222, 225,    228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264,    267, 270, 273, 276, 279, 282, 285, 288, 291, 306, 309, 318, 321,    324, 327, 330, 333, 342, 345, 348, 351, 354, 357, 360, 363, 366,    369, 384, 387, 390, 393, 396 or 399;-   (b) a polynucleotide encoding a polypeptide having the amino acid    sequence of SEQ ID NO: 6, 8, 10, 12, 18, 377, 379 or 381;-   (c) a polynucleotide capable of hybridizing to a CYP2C8 gene,    wherein said polynucleotide is having at a position corresponding to    position 411, 560, 713, 817, 824, 831, 879, 886, 1058, 1627, 1668,    1767, 1887, 1905 or 1952 (GenBank accession No: AF136830.1), at a    position corresponding to position 171 or 258 (GenBank accession No:    AF136832.1), at a position corresponding to position 122, 150, 182,    334, 339 or 378 (GenBank accession No: AF136833.1), at a position    corresponding to position 162, 163, 243 (GenBank accession No:    AF136834.2) or at position 583 (GenBank accession No:    NM_(—)000770.1), at a position corresponding to position 13 or 180    (GenBank accession No: AF136835.1), at a position corresponding to    position 116, 132, 172 or 189 (GenBank accession No: AF136836.1), at    a position corresponding to position 42 or 101 (GenBank accession    No: AF136837.1), at a position corresponding to position 309    (GenBank accession No: AF136838.1), at a position corresponding to    position 1135 (GenBank accession No: NM_(—)000770.1), at a position    corresponding to position 232 (GenBank accession No: AF136840.1), at    a position corresponding to position 206 (GenBank accession No:    AF136842.1), at a position corresponding to position 30, 87, 167,    197, 212, 221, 255 or 271 (GenBank accession No: AF136843.1), at a    position corresponding to position 118 (GenBank accession No:    AF136844.1), at a position corresponding to position 44 (GenBank    accession No: AF136845.1) of the cytochrome 2C8 gene (GenBank    accession No: GI: 13787189) a nucleotide substitution, at a position    corresponding to position 306 to 307, 1271 to 1273 or 1397 to 1398    of the CYP2C8 gene (GenBank accession No: AF136830.1), at a position    corresponding to position 329 of the CYP2C8 gene (GenBank accession    No: AF136833.1), at a position corresponding to position 87 of the    CYP2C8 gene (GenBank accession No: AF136834.2) a deletion of one or    more nucleotides or at a position corresponding to position    1785/1786 of the CYP2C8 gene (GenBank accession No: AF136830.1) or    at a position corresponding to position 180/181 of the CYP2C8 gene    (GenBank accession No: AF136833.1) an insertion of one or more    nucleotides;-   (d) a polynucleotide capable of hybridizing to a CYP2C8 gene,    wherein said polynucleotide is having at a position corresponding to    position 411, 817, 824, 831, 879, 1058, 1767 or 1887 of the CYP2C8    gene (GenBank accession No: AF136830.1) an A, at a position    corresponding to position 560 or 1668 of the CYP2C8 gene (GenBank    accession No: AF136830.1) a G, at a position corresponding to    position 713 or 886 of the CYP2C8 gene (GenBank accession No:    AF136830.1) a T, at a position corresponding to position 1627, 1905    or 1952 of the CYP2C8 gene (GenBank accession No: AF136830.1) a C,    at a position corresponding to position 258 of the CYP2C8 gene    (GenBank accession No: AF136832.1) a T, at a position corresponding    to position 171 of the CYP2C8 gene (GenBank accession No:    AF136832.1) a C, at a position corresponding to position 122, 150 or    334 of the CYP2C8 gene (GenBank accession No: AF136833.1) an A, at a    position corresponding to position 182 or 378 of the CYP2C8 gene    (GenBank accession No: AF136833.1) a C, at a position corresponding    to position 162, 163, 243 [identical to position corresponding to    position 583 of the CYP2C8 gene (GenBank accession No:    NM_(—)000770.1) of the CYP2C8 gene (GenBank accession No:    AF136834.2) an A, at a position corresponding to position 180 of the    CYP2C8 gene (GenBank accession No: AF136835.1) an A, at a position    corresponding to position 13 of the CYP2C8 gene (GenBank accession    No: AF136835.1) a G, at a position corresponding to position 116 or    132 of the CYP2C8 gene (GenBank accession No: AF136836.1) a G, at a    position corresponding to position 172 of the CYP2C8 gene (GenBank    accession No: AF136836.1) a G, at a position corresponding to    position 189 of the CYP2C8 gene (GenBank accession No: AF136836.1) a    C, at a position corresponding to position 42 or 101 of the CYP2C8    gene (GenBank accession No: AF136837.1) a G, at a position    corresponding to position 1135 of the CYP2C8 gene (GenBank accession    No: GI: 13787189) an A, at a position corresponding to position 309    of the CYP2C8 gene (GenBank accession No: AF136838.1) a T, at a    position corresponding to position 232 (GenBank accession No:    136840.1) a T, at a position corresponding to position 30 or 212 of    the CYP2C8 gene (GenBank accession No: AF136843.1) a T, at a    position corresponding to position 87 of the CYP2C8 gene (GenBank    accession No: AF136843.1) a G, at a position corresponding to    position 167 or 197 of the CYP2C8 gene (GenBank accession No:    AF136843.1) an A, at a position corresponding to position 221, 255    or 271 of the CYP2C8 gene (GenBank accession No: AF136843.1) a C, at    a position corresponding to position 118 of the CYP2C8 gene (GenBank    accession No: AF136844.1) an A, at a position corresponding to    position 44 of the CYP2C8 gene (GenBank accession No: AF136845.1) a    T;-   (e) a polynucleotide encoding a molecular CYP2C8 variant polypeptide    or fragment thereof, wherein said polypeptide comprises an amino    acid substitution at a position corresponding to any one of position    159, 181, 209, 244, 263, 274, 343 or 365 of the CYP2C8 polypeptide    (GI: 13787189); and-   (f) a polynucleotide encoding a molecular CYP2C8 variant polypeptide    or fragment thereof, wherein said polypeptide comprises an amino    acid substitution of T to P at position corresponding to position    159 (frameshift), V to I at a position corresponding to position    181, N to S at a position corresponding to position 209, I to V at a    position corresponding to position 244, F to L at a position    corresponding to position 263, E to Stop at a position corresponding    to position 274, G to S at a position corresponding to position 365    or S to I at a position corresponding to position 343 of the CYP2C8    polypeptide (GenBank accession No: Gl: 13787189).

In the context of the present invention the term “polynucleotides” orthe term “polypeptides” refers to different variants of a polynucleotideor polypeptide. Said variants comprise a reference or wild type sequenceof the polynucleotides or polypeptides of the invention as well asvariants which differ therefrom in structure or composition. Referenceor wild type sequences for the polynucleotides are GenBank accession No:NM_(—)000770.1 for mRNA. Reference or wild type sequence for thepolynucleotide of the invention is for the 5′UTR: GenBank accession No:AF136830.1; for exon 1: GenBank accession No: AF136831.1; for exon 2:GenBank accession No: AF136832.1 and AF136833.1; for exon 3: GenBankaccession No: AF136833.1; for exon 4: GenBank accession No: AF136834.2and AF136835.1; for exon 5: GenBank accession No: AF136836.1 andAF136837.1; for exon 6: GenBank accession No: AF136838.1 and AF136839.1;for exon 7: GenBank accession No: AF136840.1 and AF136841.1; for exon 8:GenBank accession No: AF136842.1 and AF136843.1; for exon 9/3′UTR:GenBank accession No: AF136844.1 and AF136845.1; partly in combinationwith NM_(—)000770.1 (mRNA). Reference or wild type sequence for thepolypeptide of the CYP2C8 gene is GenBank accession No: GI: 13787189. Inthe context of the present invention the term “5′UTR” refers to theuntranslated region 5′ to the ATG start codon including the 5′-upstreamregion encompassing the promoter. The term “3′UTR” refers to theuntranslated region 3′ to the Stop codon.

The differences in structure or composition usually occur by way ofnucleotide or amino acid substitution(s), addition(s) and/ordeletion(s). Preferred substitution in accordance with the presentinvention are a T to G substitution at a position corresponding toposition 1668 (GenBank accession No: AF136830.1), a G to A substitutionat a position corresponding to position 831 (GenBank accession No:AF136830.1), a G to T substitution at a position corresponding toposition 309 (GenBank accession No: AF136838.1) and 232 (GenBankaccession No: AF136840.1) of the CYP2C8 gene. Preferred deletions inaccordance with the invention are an AT deletion at a positioncorresponding to position 1397 to 1398 (GenBank accession No:AF136830.1) and a deletion of at least one A at a position correspondingto position 329 (GenBank accession No: AF136833.1) of the CYP2C8 gene.

In accordance with the present invention it has also been found that adeletion of the nucleotide A at a position corresponding to position 329(GenBank accession No: AF136833.1) of the CYP2C8 gene leads to analtered C-terminus of the protein encoding a CYP2C8 polypeptide whereinsaid polypeptide comprises an amino acid substitution of T to P at aposition corresponding to position 159 of the CYP2C8 polypeptide(GenBank accession No: GI: 13787189). In accordance with the presentinvention it has also been found that a substitution of a G to a T at aposition corresponding to position 309 (GenBank accession No:AF136838.1) of the CYP2C8 gene leads to a polypeptide wherein saidpolypeptide comprises an amino acid substitution of E to a prematuretermination (stop) at a position corresponding to position 274 of theCYP2C8 polypeptide (GenBank accession No: GI: 13787189) and asubstitution of the nucleotide G to an A at a position corresponding toposition 1135 (GenBank accession No: NM_(—)000770.1) of the CYP2C8 geneleads to a polypeptide wherein said polypeptide comprises an amino acidsubstitution of G to S at a position corresponding to position 365 ofthe CYP2C8 polypeptide (GenBank accession No: GI: 13787189). This willalter the structure or confirmation of the protein and will abolish theactivity of the drug metabolizing enzyme.

Preferably, said nucleotide substitution(s), addition(s) or deletion(s)comprised by the present invention result(s) in one or more changes ofthe corresponding amino acid(s) of the polypeptides of the invention.

The variant polynucleotides and polypeptides also comprise fragments ofsaid polynucleotides or polypeptides of the invention. Thepolynucleotides and polypeptides as well as the aforementioned fragmentsthereof of the present invention are characterized as being associatedwith a CYP2C8 dysfunction or dysregulation comprising, e.g.,insufficient and/or altered metabolism. Said dysfunctions ordysregulations referred to in the present invention cause a disease ordisorder or a prevalence for said disease or disorder. Preferably, aswill be discussed below in detail, said disease is a deficiency in themetabolism of certain drugs which are metabolized by CYP2C8, e.g. TAXOL(paclitaxel), Verapamil, or any other disease caused by a dysfunction ordysregulation due to a polynucleotide or polypeptides of the invention,also referred to as CYP2C8 gene associated diseases in the following.

The term “hybridizing” as used herein refers to polynucleotides whichare capable of hybridizing to the polynucleotides of the invention orparts thereof which are associated with a CYP2C8 dysfunction ordysregulation. Thus, said hybridizing polynucleotides are alsoassociated with said dysfunctions and dysregulations. Preferably, saidpolynucleotides capable of hybridizing to the polynucleotides of theinvention or parts thereof which are associated with CYP2C8 dysfunctionsor dysregulations are at least 70%, at least 80%, at least 95% or atleast 100% identical to the polynucleotides of the invention or partsthereof which are associated with CYP2C8 dysfunctions or dysregulations.Therefore, said polynucleotides may be useful as probes in Northern orSouthern Blot analysis of RNA or DNA preparations, respectively, or canbe used as oligonucleotide primers in PCR analysis dependent on theirrespective size. Also comprised by the invention are hybridizingpolynucleotides which are useful for analyzing DNA-Protein interactionsvia, e.g., electrophoretic mobility shift analysis (EMSA). Preferably,said hybridizing polynucleotides comprise at least 10, more preferablyat least 15 nucleotides in length while a hybridizing polynucleotide ofthe present invention to be used as a probe preferably comprises atleast 100, more preferably at least 200, or most preferably at least 500nucleotides in length.

It is well known in the art how to perform hybridization experimentswith nucleic acid molecules, i.e. the person skilled in the art knowswhat hybridization conditions s/he has to use in accordance with thepresent invention. Such hybridization conditions are referred to instandard text books such as Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y. Preferred in accordance with thepresent inventions are polynucleotides which are capable of hybridizingto the polynucleotides of the invention or parts thereof which areassociated with a CYP2C8 dysfunction or dysregulation under stringenthybridization conditions, i.e. which do not cross hybridize to unrelatedpolynucleotides such as polynucleotides encoding a polypeptide differentfrom the CYP2C8 polypeptides of the invention.

The term “corresponding” as used herein means that a position is notonly determined by the number of the preceding nucleotides and aminoacids, respectively. The position of a given nucleotide or amino acid inaccordance with the present invention which may be deleted, substitutedor comprise one or more additional nucleotide(s) may vary due todeletions or additional nucleotides or amino acids elsewhere in the geneor the polypeptide. Thus, under a “corresponding position” in accordancewith the present invention it is to be understood that nucleotides oramino acids may differ in the indicated number but may still havesimilar neighboring nucleotides or amino acids. Said nucleotides oramino acids which may be exchanged, deleted or comprise additionalnucleotides or amino acids are also comprised by the term “correspondingposition”. Said nucleotides or amino acids may for instance togetherwith their neighbors form sequences which may be involved in theregulation of gene expression, stability of the corresponding RNA or RNAediting, as well as encode functional domains or motifs of the proteinof the invention.

By, e.g., “position 1271 to 1273” it is meant that said polynucleotidecomprises one or more deleted nucleotides which are deleted betweenpositions 1271 and position 1273 of the corresponding wild type versionof said polynucleotide. The same applies mutatis mutandis to all otherposition numbers referred to in the above embodiment which are draftedin the same format.

By, e.g., “position 180/181” it is meant that said polynucleotidecomprises one or more additional nucleotide(s) which are insertedbetween positions 180 and position 181 of the corresponding wild typeversion of said polynucleotide. The same applies mutatis mutandis to allother position numbers referred to in the above embodiment which aredrafted in the same format, i.e. two consecutive position numbersseparated by a slash (/).

In accordance with the present invention, the mode and populationdistribution of genetic variations in the CYP2C8 gene has been analyzedby sequence analysis of relevant regions of the human said gene frommany different individuals. It is a well known fact that genomic DNA ofindividuals, which harbor the individual genetic makeup of all genes,including the CYP2C8 gene, can easily be purified from individual bloodsamples. These individual DNA samples are then used for the analysis ofthe sequence composition of the alleles of the CYP2C8 gene that arepresent in the individual which provided the blood sample. The sequenceanalysis was carried out by PCR amplification of relevant regions ofsaid genes, subsequent purification of the PCR products, followed byautomated DNA sequencing with established methods (e.g. ABI dyeterminator cycle sequencing).

One important parameter that had to be considered in the attempt todetermine the individual genotypes and identify novel variants of theCYP2C8 gene by direct DNA-sequencing of PCR-products from human bloodgenomic DNA is the fact that each human harbors (usually, with very fewabnormal exceptions) two gene copies of each autosomal gene (diploidy).Because of that, great care had to be taken in the evaluation of thesequences to be able to identify unambiguously not only homozygoussequence variations but also heterozygous variations. The details of thedifferent steps in the identification and characterization of novelpolymorphisms in the CYP2C8 gene (homozygous and heterozygous) aredescribed in the examples below.

Over the past 20 years, genetic heterogeneity has been increasinglyrecognized as a significant source of variation in drug response. Manyscientific communications (Meyer, Ann. Rev. Pharmacol. Toxicol. 37(1997), 269-296 and West, J. Clin. Pharmacol. 37 (1997), 635-648) haveclearly shown that some drugs work better or may even be highly toxic insome patients than in others and that these variations in patient'sresponses to drugs can be related to molecular basis. This“pharmacogenomic” concept spots correlations between responses to drugsand genetic profiles of patient's (Marshall, Nature Biotechnology, 15(1997), 954-957; Marshall, Nature Biotechnology, 15 (1997), 1249-1252).In this context of population variability with regard to drug therapy,pharmacogenomics has been proposed as a tool useful in theidentification and selection of patients which can respond to aparticular drug without side effects. This identification/selection canbe based upon molecular diagnosis of genetic polymorphisms by genotypingDNA from leukocytes in the blood of patient, for example, andcharacterization of disease (Bertz, Clin. Pharmacokinet. 32 (1997),210-256; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103).For the founders of health care, such as health maintenanceorganizations in the US and government public health services in manyEuropean countries, this pharmacogenomics approach can represent a wayof both improving health care and reducing overheads because there is alarge cost to unnecessary drugs, ineffective drugs and drugs with sideeffects.

The mutations in the variant genes of the invention sometime result inamino acid deletion(s), insertion(s) and in particular insubstitution(s) either alone or in combination. It is of course alsopossible to genetically engineer such mutations in wild type genes orother mutant forms. Methods for introducing such modifications in theDNA sequence of said genes are well known to the person skilled in theart; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y.

For the investigation of the nature of the alterations in the amino acidsequence of the polypeptides of the invention may be used such asBRASMOL that are obtainable from the Internet. Furthermore, foldingsimulations and computer redesign of structural motifs can be performedusing other appropriate computer programs (Olszewski, Proteins 25(1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).Computers can be used for the conformational and energetic analysis ofdetailed protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012;Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). These analysis can beused for the identification of the influence of a particular mutation onmetabolizing, binding and/or transport of drugs.

Usually, said amino acid deletion, addition or substitution in the aminoacid sequence of the protein encoded by the polynucleotide of theinvention is due to one or more nucleotide substitution, insertion ordeletion, or any combinations thereof. Preferably said nucleotidesubstitution, insertion or deletion may result in an amino acidsubstitution of F to L at position corresponding to position 263 of theCYP2C8 polypeptide (GenBank accession No: GI: 13787189). Thepolypeptides encoded by the polynucleotides of the invention havealtered biological or immunological properties due to the mutationsreferred to in accordance with the present invention. Examples for saidaltered properties are stability of the polypeptides which may beeffected or an altered substrate specificity or even a complete loss ofthe capability of metabolizing certain drugs.

The mutations in the CYP2C8 gene detected in accordance with the presentinvention are listed in Table 2. The methods of the mutation analysisfollowed standard protocols and are described in detail in the Examples.In general such methods are to be used in accordance with the presentinvention for evaluating the phenotypic spectrum as well as theoverlapping clinical characteristics of diseases or conditions relatedto dysfunctions or dysregulations and diseases related to the poor orextensive metabolism (PM or EM) certain drugs.

Advantageously, the characterization of said mutants may form the basisof the development of a diagnostic assay that is able to predict apatients efficacy to metabolize a drug for instance in anticancertreatment (TAXOL (paclitaxel)) or cardiovascular deficiencies(verapamil). Said methods encompass for example haplotype analysis,single-strand conformation polymorphism analysis (SSCA), PCR and directsequencing. On the basis of thorough clinical characterization of manypatients the phenotypes can then be correlated to these mutations.

Also comprised by the polynucleotides referred to in the presentinvention are polynucleotides which comprise at least two of thepolynucleotides specified herein above, i.e. polynucleotides having anucleotide sequence which contains all four mutations comprised by theabove polynucleotides or listed in Table 2 below (haplotype: positions831, 1397 to 1398 of GenBank accession No: AF136830.1, position 270 ofGenBank accession No: AF136833.1, and position 206 of (GenBank accessionNo: AF136842.1). In accordance with the present invention it is alsopreferred to detect only one of the above mentioned polymorphisms of thehaplotype, said one polymorphism being indicative for the presence ofthe other polymorphisms of the haplotype. Thus, in order to detect thepresence of the above mentioned haplotype it is sufficient to determinethe presence of any one of the polymorphisms comprised by saidhaplotype. Moreover, the polynucleotides referred to above allow thestudy of synergistic effects of said mutations in the CYP2C8 gene and/ora polypeptide encoded by said polynucleotide on the pharmacologicalprofile of drugs in patients who bear such mutant forms of the gene orsimilar mutant forms that can be mimicked by the above describedproteins. It is expected that the analysis of said synergistic effectsprovides deeper insights into the onset of CYP2C8 dysfunctions ordysregulations or diseases related to altered drug transport asdescribed supra. From said deeper insight the development of diagnosticand pharmaceutical compositions related to CYP2C8 dysfunctions ordysregulations or diseases related to impaired drug metabolism willgreatly benefit.

As is evident to the person skilled in the art, the genetic knowledgededuced from the present invention can now be used to exactly andreliably characterize the genotype of a patient. Advantageously,diseases or a prevalence for a disease which are associated with CYP2C8dysfunction or dysregulation, e.g. diseases associated with arachidonicacid metabolism referred to herein can be predicted and preventive ortherapeutical measures can be applied accordingly. Moreover inaccordance with the foregoing, in cases where a given drug takes anunusual effect, a suitable individual therapy can be designed based onthe knowledge of the individual genetic makeup of a subject with respectto the polynucleotides of the invention and improved therapeutics can bedeveloped as will be further discussed below.

In general, the CYP2C8 “status”, defined by the expression level andactivity of the CYP2C8 protein, can be variable in normal tissue, due togenetic variations/polymorphisms. The identification of polymorphismsassociated with altered CYP2C8 expression and/or activity is importantfor the prediction of drug metabolism and subsequently for theprediction of therapy outcome, including side effects of medications.Therefore, analysis of CYP2C8 variations indicative of CYP2C8 function,is a valuable tool for therapy with drugs, which are substrates ofCYP2C8 and has, thanks to the present invention, now become possible.

In line with the foregoing, preferably, the polynucleotide of thepresent invention is associated with an incompatibility or a diseaserelated to arachidonic acid metabolism, cancer or cardiovasculardiseases.

The term “cancer” used herein is very well known and characterized inthe art. Several variants of cancer exist and are comprised by said termas meant in accordance with the invention. For a detailed list ofsymptoms which are indicative for cancer it is referred to text bookknowledge, e.g. Pschyrembel. The term “cardiovascular disease” as usedherein refers to those diseases known in the art and described in detailin standard text books, such as Pschyrembel or Stadman. Examples forcardiovascular diseases are hypertension or atherosclerosis. Theinefficacy or complete loss to epoxidate arachidonoic acid is referredto as disease of the arachidonic acid metabolism.

In a further embodiment the present invention relates to apolynucleotide which is DNA or RNA.

The polynucleotide of the invention may be, e.g., DNA, cDNA, genomicDNA, RNA or synthetically produced DNA or RNA or a recombinantlyproduced chimeric nucleic acid molecule comprising any of thosepolynucleotides either alone or in combination. Preferably saidpolynucleotide is part of a vector, particularly plasmids, cosmids,viruses and bacteriophages used conventionally in genetic engineeringthat comprise a polynucleotide of the invention. Such vectors maycomprise further genes such as marker genes which allow for theselection of said vector in a suitable host cell and under suitableconditions.

The invention furthermore relates to a gene comprising thepolynucleotide of the invention.

It is well known in the art that genes comprise structural elementswhich encode an amino acid sequence as well as regulatory elements whichare involved in the regulation of the expression of said genes.Structural elements are represented by exons which may either encode anamino acid sequence or which may encode for RNA which is not encoding anamino acid sequence but is nevertheless involved in RNA function, e.g.by regulating the stability of the RNA or the nuclear export of the RNA.

Regulatory elements of a gene may comprise promoter elements or enhancerelements both of which could be involved in transcriptional control ofgene expression. It is very well known in the art that a promoter is tobe found upstream of the structural elements of a gene. Regulatoryelements such as enhancer elements, however, can be found distributedover the entire locus of a gene. Said elements could be reside, e.g., inintrons, regions of genomic DNA which separate the exons of a gene.Promoter or enhancer elements correspond to polynucleotide fragmentswhich are capable of attracting or binding polypeptides involved in theregulation of the gene comprising said promoter or enhancer elements.For example, polypeptides involved in regulation of said gene comprisethe so called transcription factors. Said introns may comprise furtherregulatory elements which are required for proper gene expression.Introns are usually transcribed together with the exons of a generesulting in a nascent RNA transcript which contains both, exon andintron sequences. The intron encoded RNA sequences are usually removedby a process known as RNA splicing. However, said process also requiresregulatory sequences present on a RNA transcript said regulatorysequences may be encoded by the introns.

In addition, besides their function in transcriptional control andcontrol of proper RNA processing and/or stability, regulatory elementsof a gene could be also involved in the control of genetic stability ofa gene locus. Said elements control, e.g., recombination events or serveto maintain a certain structure of the DNA or the arrangement of DNA ina chromosome.

Therefore, single nucleotide polymorphisms can occur in exons of a genewhich encode an amino acid sequence as discussed supra as well as inregulatory regions which are involved in the above discussed process.The analysis of the nucleotide sequence of a gene locus in its entiretyincluding, e.g., introns is in light of the above desirable. Thepolymorphisms comprised by the polynucleotides of the present inventioncan influence the expression level of CYP2C8 protein via mechanismsinvolving enhanced or reduced transcription of the CYP2C8 gene,stabilization of the gene's RNA transcripts and alteration of theprocessing of the primary RNA transcripts.

Therefore, in a furthermore preferred embodiment of the gene of theinvention a nucleotide deletion, addition and/or substitution results inaltered expression of the variant gene compared to the correspondingwild type gene.

In another embodiment the present invention relates to a vectorcomprising the polynucleotide of the invention or the gene of theinvention.

Said vector may be, for example, a phage, plasmid, viral or retroviralvector. Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host/cells.

The polynucleotides or genes of the invention may be joined to a vectorcontaining selectable markers for propagation in a host. Generally, aplasmid vector is introduced in a precipitate such as a calciumphosphate precipitate, or in a complex with a charged lipid or incarbon-based clusters. Should the vector be a virus, it may be packagedin vitro using an appropriate packaging cell line prior to applicationto host cells.

In a more preferred embodiment of the vector of the invention thepolynucleotide is operatively linked to expression control sequencesallowing expression in prokaryotic or eukaryotic cells or isolatedfractions thereof.

Expression of said polynucleotide comprises transcription of thepolynucleotide, preferably into a translatable mRNA. Regulatory elementsensuring expression in eukaryotic cells, preferably mammalian cells, arewell known to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers. Possible regulatory elementspermitting expression in prokaryotic host cells comprise, e.g., the sac,trp or tac promoter in E. coli, and examples for regulatory elementspermitting expression in eukaryotic host cells are the AOX1 or GAL1promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells. Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pSPORT1 (GIBCO BRL). Preferably, said vector is anexpression vector and/or a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotides or vector of the inventioninto targeted cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectors;see, for example, the techniques described in Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Alternatively, thepolynucleotides and vectors of the invention can be reconstituted intoliposomes for delivery to target cells.

The term “isolated fractions thereof” refers to fractions of eukaryoticor prokaryotic cells or tissues which are capable of transcribing ortranscribing and translating RNA from the vector of the invention. Saidfractions comprise proteins which are required for transcription of RNAor transcription of RNA and translation of said RNA into a polypeptide.Said isolated fractions may be, e.g., nuclear and cytoplasmic fractionsof eukaryotic cells such as of reticulocytes.

The present invention furthermore relates to a host cell geneticallyengineered with the polynucleotide of the invention, the gene of theinvention or the vector of the invention.

Said host cell may be a prokaryotic or eukaryotic cell; see supra. Thepolynucleotide or vector of the invention which is present in the hostcell may either be integrated into the genome of the host cell or it maybe maintained extrachromosomally. In this respect, it is also to beunderstood that the recombinant DNA molecule of the invention can beused for “gene targeting” and/or “gene replacement”, for restoring amutant gene or for creating a mutant gene via homologous recombination;see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990),47124716; Joyner, Gene Targeting, A Practical Approach, OxfordUniversity Press.

The host cell can be any prokaryotic or eukaryotic cell, such as abacterial, insect, fungal, plant, animal, mammalian or, preferably,human cell. Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a polynucleotide for the expression of avariant polypeptide of the invention. Prokaryotic hosts may include gramnegative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Apolynucleotide coding for a mutant form of variant polypeptides of theinvention can be used to transform or transfect the host using any ofthe techniques commonly known to those of ordinary skill in the art.Methods for preparing fused, operably linked genes and expressing themin bacteria or animal cells are well-known in the art (Sambrook, supra).The genetic constructs and methods described therein can be utilized forexpression of variant polypeptides of the invention in, e.g.,prokaryotic hosts. In general, expression vectors containing promotersequences which facilitate the efficient transcription of the insertedpolynucleotide are used in connection with the host. The expressionvector typically contains an origin of replication, a promoter, and aterminator, as well as specific genes which are capable of providingphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be grown in fermentors and cultured according totechniques known in the art to achieve optimal cell growth. The proteinsof the invention can then be isolated from the grown medium, cellularlysates, or cellular membrane fractions. The isolation and purificationof the microbially or otherwise expressed polypeptides of the inventionmay be by any conventional means such as, for example, preparativechromatographic separations and immunological separations such as thoseinvolving the use of monoclonal or polyclonal antibodies.

Thus, in a further embodiment the invention relates to a method forproducing a molecular variant CYP2CB polypeptide or fragment thereofcomprising culturing the above described host cell; and recovering saidprotein or fragment from the culture.

In another embodiment the present invention relates to a method forproducing cells capable of expressing a molecular variant CYP2C8polypeptide comprising genetically engineering cells with thepolynucleotide of the invention, the gene of the invention or the vectorof the invention.

The cells obtainable by the method of the invention can be used, forexample, to test drugs according to the methods described in D. L.Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab manual, CSH Press1998. Furthermore, the cells can be used to study known drugs andunknown derivatives thereof for their ability to complement thedeficiency caused by mutations in the CYP2C8 gene. For these embodimentsthe host cells preferably lack a wild type allele, preferably bothalleles of the CYP2C8 gene and/or have at least one mutated fromthereof. Ideally, the gene comprising an allele as comprised by thepolynucleotides of the invention could be introduced into the wild typelocus by homologous replacement. Alternatively, strong overexpression ofa mutated allele over the normal allele and comparison with arecombinant cell line overexpressing the normal allele at a similarlevel may be used as a screening and analysis system. The cellsobtainable by the above-described method may also be used for thescreening methods referred to herein below.

Furthermore, the invention relates to a polypeptide or fragment thereofencoded by the polynucleotide of the invention, the gene of theinvention or obtainable by the method described above or from cellsproduced by the method described above. In this context it is alsounderstood that the variant polypeptide of the invention can be furthermodified by conventional methods known in the art. By providing saidvariant proteins according to the present invention it is also possibleto determine the portions relevant for their biological activity orinhibition of the same. The terms “polypeptide” and “protein” as usedherein are exchangeable. Moreover, what is comprised by said terms isstandard textbook knowledge.

The present invention furthermore relates to an antibody which bindsspecifically to the polypeptide of the invention.

Advantageously, the antibody specifically recognizes or binds an epitopecontaining one or more amino acid substitution(s) as defined above.Antibodies against the variant polypeptides of the invention can beprepared by well known methods using a purified protein according to theinvention or a (synthetic) fragment derived therefrom as an antigen.Monoclonal antibodies can be prepared, for example, by the techniques asoriginally described in Köhler and Milstein, Nature 256 (1975), 495, andGalfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mousemyeloma cells to spleen cells derived from immunized mammals. In apreferred embodiment of the invention, said antibody is a monoclonalantibody, a polyclonal antibody, a single chain antibody, human orhumanized antibody, primatized, chimerized or fragment thereof thatspecifically binds said peptide or polypeptide also including bispecificantibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFvfragments etc., or a chemically modified derivative of any of these.Furthermore, antibodies or fragments thereof to the aforementionedpolypeptides can be obtained by using methods which are described, e.g.,in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of the variant polypeptidesof the invention as well as for the monitoring of the presence of saidvariant polypeptides, for example, in recombinant organisms, and for theidentification of compounds interacting with the proteins according tothe invention. For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies which bind to an epitope of the protein of the invention(Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol. Methods 183 (1995), 7-13).

In a preferred embodiment the antibody of the present inventionspecifically recognizes an epitope containing one or more amino acidsubstitution(s) resulting from a nucleotide exchange as defined supra.

Antibodies which specifically recognize modified amino acids such asphospho-Tyrosine residues are well known in the art. Similarly, inaccordance with the present invention antibodies which specificallyrecognize even a single amino acid exchange in an epitope may begenerated by the well known methods described supra.

In light of the foregoing, in a more preferred embodiment the antibodyof the present invention is monoclonal or polyclonal.

The invention also relates to a transgenic non-human animal comprisingat least one polynucleotide of the invention, the gene of the inventionor the vector of the invention as described supra.

The present invention also encompasses a method for the production of atransgenic non-human animal comprising introduction of a polynucleotideor vector of the invention into a germ cell, an embryonic cell, stemcell or an egg or a cell derived therefrom. The non-human animal can beused in accordance with the method of the invention described below andmay be a non-transgenic healthy animal, or may have a disease ordisorder, preferably a disease caused by at least one mutation in thegene of the invention. Such transgenic animals are well suited for,e.g., pharmacological studies of drugs in connection with variant formsof the above described variant polypeptides since these polypeptides orat least their functional domains are conserved between species inhigher eukaryotes, particularly in mammals. Production of transgenicembryos and screening of those can be performed, e.g., as described byA. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), OxfordUniversity Press. The DNA of the embryos can be analyzed using, e.g.,Southern blots with an appropriate probe or based on PCR techniques. Atransgenic non-human animal in accordance with the invention may be atransgenic mouse, rat, hamster, dog, monkey, rabbit, pig, frog, nematodesuch as Caenorhabditis elegans, fruitfly such as Drosophila melanogasteror fish such as torpedo fish or zebrafish comprising a polynucleotide orvector of the invention or obtained by the method described above,preferably wherein said polynucleotide or vector is stably integratedinto the genome of said non-human animal, preferably such that thepresence of said polynucleotide or vector leads to the expression of thevariant polypeptide of the invention. It may comprise one or severalcopies of the same or different polynucleotides or genes of theinvention. This animal has numerous utilities, including as a researchmodel for cardiovascular research and therefore, presents a novel andvaluable animal in the development of therapies, treatment, etc. fordiseases caused by cardiovascular diseases. Accordingly, in thisinstance, the mammal is preferably a laboratory animal such as a mouseor rat.

Thus, in a preferred embodiment the transgenic non-human animal of theinvention is a mouse, a rat or a zebrafish.

Numerous reports revealed that said animals are particularly well suitedas model organisms for the investigation of the drug metabolism and itsdeficiencies or cancer. Advantageously, transgenic animals can be easilycreated using said model organisms, due to the availability of varioussuitable techniques well known in the art.

The invention also relates to a solid support comprising one or aplurality of the polynucleotide, the gene, the vector, the polypeptide,the antibody or the host cell of the invention in immobilized form.

The term “solid support” as used herein refers to a flexible ornon-flexible support that is suitable for carrying said immobilizedtargets. Said solid support may be homogenous or inhomogeneous. Forexample, said solid support may consist of different materials havingthe same or different properties with respect to flexibility andimmobilization, for instance, or said solid support may consist of onematerial exhibiting a plurality of properties also comprisingflexibility and immobilization properties. Said solid support maycomprise glass-, polypropylene- or silicon-chips, membranesoligonucleotide-conjugated beads or bead arrays.

The term “immobilized” means that the molecular species of interest isfixed to a solid support, preferably covalently linked thereto. Thiscovalent linkage can be achieved by different means depending on themolecular nature of the molecular species. Moreover, the molecularspecies may be also fixed on the solid support by electrostatic forces,hydrophobic or hydrophilic interactions or Van-der-Waals forces. Theabove described physico-chemical interactions typically occur ininteractions between molecules. For example, biotinylated polypeptidesmay be fixed on a avidin-coated solid support due to interactions of theabove described types. Further, polypeptides such as antibodies, may befixed on an antibody coated solid support. Moreover, the immobilizationis dependent on the chemical properties of the solid support. Forexample, the nucleic acid molecules can be immobilized on a membrane bystandard techniques such as UV-crosslinking or heat.

In a preferred embodiment of the invention said solid support is amembrane, a glass- or polypropylene- or silicon-chip, areoligonucleotide-conjugated beads or a bead array, which is assembled onan optical filter substrate.

Moreover, the present invention relates to an in vitro method foridentifying a polymorphism said method comprising the steps of:

-   (a) isolating a polynucleotide or the gene of the invention from a    plurality of subgroups of individuals, wherein one subgroup has no    prevalence for a CYP2C8 associated disease and at least one or more    further subgroup(s) do have prevalence for a CYP2C8 associated    disease; and-   (b) identifying a polymorphism by comparing the nucleic acid    sequence of said polynucleotide or said gene of said one subgroup    having no prevalence for a CYP2C8 associated disease with said at    least one or more further subgroup(s) having a prevalence for a    CYP2C8 associated disease.

The term “prevalence” as used herein means that individuals are besusceptible for one or more disease(s) which are associated with CYP2C8dysfunction or dysregulation or could already have one or more of saiddisease(s). Thereby, one CYP2C8 associated disease can be used todetermine the susceptibility for another CYP2C8 associated disease.Moreover, symptoms which are indicative for a prevalence for developingof a disease are very well known in the art and have been sufficientlydescribed in standard textbooks such as Pschyrembel.

Advantageously, polymorphisms according to the present invention whichare associated with CYP2C8 dysfunction or dysregulation or one or moredisease(s) based thereon should be enriched in subgroups of individualswhich have a prevalence for said diseases versus subgroups which have noprevalence for said diseases. Thus, the above described method allowsthe rapid and reliable detection of polymorphism which are indicativefor one or more CYP2C8 associated disease(s) or a susceptibilitytherefor. Advantageously, due to the phenotypic preselection a largenumber of individuals having no prevalence might be screened forpolymorphisms in general. Thereby, a reference sequences comprisingpolymorphisms which do not correlate to one or more CYP2C8 associateddisease(s) can be obtained. Based on said reference sequences it ispossible to efficiently and reliably determine the relevantpolymorphisms.

In a further embodiment the present invention relates to a method foridentifying and obtaining a pro-drug or a drug capable of modulating theactivity of a molecular variant of a CYP2C8 polypeptide comprising thesteps of:

-   (a) contacting the polypeptide, the solid support of the invention,    a cell expressing a molecular variant gene comprising a    polynucleotide of the invention, the gene or the vector of the    invention in the presence of components capable of providing a    detectable signal in response to drug activity with a compound to be    screened for pro-drug or drug activity; and-   (b) detecting the presence or absence of a signal or increase or    decrease of a signal generated from the pro-drug or the drug    activity, wherein the absence, presence, increase or decrease of the    signal is indicative for a putative pro-drug or drug.

The term “compound” in a method of the invention includes a singlesubstance or a plurality of substances which may or may not beidentical.

Said compound(s) may be chemically synthesized or produced via microbialfermentation but can also be comprised in, for example, samples, e.g.,cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compounds may be known in the art but hitherto notknown to be useful as an inhibitor, respectively. The plurality ofcompounds may be, e.g., added to the culture medium or injected into acell or non-human animal of the invention.

If a sample containing (a) compound(s) is identified in the method ofthe invention, then it is either possible to isolate the compound fromthe original sample identified as containing the compound, in questionor one can further subdivide the original sample, for example, if itconsists of a plurality of different compounds, so as to reduce thenumber of different substances per sample and repeat the method with thesubdivisions of the original sample. It can then be determined whethersaid sample or compound displays the desired properties, for example, bythe methods described herein or in the literature (Spector et al., Cellsmanual; see supra). Depending on the complexity of the samples, thesteps described above can be performed several times, preferably untilthe sample identified according to the method of the invention onlycomprises a limited number of or only one substance(s). Preferably saidsample comprises substances of similar chemical and/or physicalproperties, and most preferably said substances are identical. Themethods of the present invention can be easily performed and designed bythe person skilled in the art, for example in accordance with other cellbased assays described in the prior art or by using and modifying themethods as described herein. Furthermore, the person skilled in the artwill readily recognize which further compounds may be used in order toperform the methods of the invention, for example, enzymes, ifnecessary, that convert a certain compound into a precursor. Suchadaptation of the method of the invention is well within the skill ofthe person skilled in the art and can be performed without undueexperimentation.

Compounds which can be used in accordance with the present inventioninclude peptides, proteins, nucleic acids, antibodies, small organiccompounds, ligands, peptidomimetics, PNAs and the like. Said compoundsmay act as agonists or antagonists of the invention. Said compounds canalso be functional derivatives or analogues of known drugs. Methods forthe preparation of chemical derivatives and analogues are well known tothose skilled in the art and are described in, for example, Beilstein,Handbook of Organic Chemistry, Springer edition New York Inc., 175 FifthAvenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, NewYork, USA. Furthermore, said derivatives and analogues can be tested fortheir effects according to methods known in the art or as described.Furthermore, peptide mimetics and/or computer aided design ofappropriate drug derivatives and analogues can be used, for example,according to the methods described below. Such analogs comprisemolecules that may have the basis structure of known CYP2C8 substrates,inhibitors and/or modulators.

Appropriate computer programs can be used for the identification ofinteractive sites of a putative inhibitor and the polypeptides of theinvention by computer assistant searches for complementary structuralmotifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriatecomputer systems for the computer aided design of protein and peptidesare described in the prior art, for example, in Berry, Biochem. Soc.Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987),1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained fromthe above-described computer analysis can be used in combination withthe method of the invention for, e.g., optimizing known inhibitors,analogs, antagonists or agonists. Appropriate peptidomimetics and otherinhibitors can also be identified by the synthesis of peptidomimeticcombinatorial libraries through successive chemical modification andtesting the resulting compounds, e.g., according to the methodsdescribed herein. Methods for the generation and use of peptidomimeticcombinatorial libraries are described in the prior art, for example inOstresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/orcrystallographic structure of said compounds and the polypeptides of theinvention can be used for the design of peptidomimetic drugs (Rose,Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4(1996), 1545-1558). It is very well known how to obtain said compounds,e.g. by chemical or biochemical standard techniques. Thus, alsocomprised by the method of the invention are means of making orproducing said compounds. In summary, the present invention providesmethods for identifying and obtaining compounds which can be used inspecific doses for the treatment of specific forms of CYP2C8 associateddiseases.

The above definitions apply mutatis mutandis to all of the methodsdescribed in the following.

In a further embodiment the present invention relates to a method foridentifying and obtaining an inhibitor of the activity of a molecularvariant of a CYP2C8 polypeptide comprising the steps of:

-   (a) contacting the protein, the solid support of the invention or a    cell expressing a molecular variant gene comprising a polynucleotide    or the gene or the vector of the invention in the presence of    components capable of providing a detectable signal in response to    drug activity with a compound to be screened for inhibiting    activity; and-   (b) detecting the presence or absence of a signal or increase or    decrease of a signal generated from the inhibiting activity, wherein    the absence or decrease of the signal is indicative for a putative    inhibitor.

In a preferred embodiment of the method of the invention said cell is acell, obtained by the method of the invention or can be obtained fromthe transgenic non-human animal as described supra.

In a still further embodiment the present invention relates to a methodof identifying and obtaining a pro-drug or drug capable of modulatingthe activity of a molecular variant of a CYP2C8 polypeptide comprisingthe steps of:

-   (a) contacting the host cell, the cell obtained by the method of the    invention, the polypeptide or the solid support of the invention    with the first molecule known to be bound by a CYP2C8 polypeptide to    form a first complex of said polypeptide and said first molecule;-   (b) contacting said first complex with a compound to be screened,    and-   (c) measuring whether said compound displaces said first molecule    from said first complex.

Advantageously, in said method said measuring step comprises measuringthe formation of a second complex of said protein and said inhibitorcandidate. Preferably, said measuring step comprises measuring theamount of said first molecule that is not bound to said protein.

In a particularly preferred embodiment of the above-described method ofsaid first molecule is a agonist or antagonist or a substrate and/or ainhibitor and/or a modulator of the polypeptide of the invention, e.g.,with a radioactive or fluorescent label.

In a still another embodiment the present invention relates to a methodof identifying and obtaining an inhibitor capable of modulating theactivity of a molecular variant of a CYP2C8 polypeptide comprising thesteps of:

-   (a) contacting the host cell or the cell obtained by the method of    the invention, the protein or the solid support of the invention    with the first molecule known to be bound by the CYP2C8 polypeptide    to form a first complex of said protein and said first molecule;-   (b) contacting said first complex with a compound to be screened,    and-   (c) measuring whether said compound displaces said first molecule    from said first complex.

In a preferred embodiment of the method of the invention said measuringstep comprises measuring the formation of a second complex of saidprotein and said compound.

In another preferred embodiment of the method of the invention saidmeasuring step comprises measuring the amount of said first moleculethat is not bound to said protein.

In a more preferred embodiment of the method of the invention said firstmolecule is labeled.

The invention furthermore relates to a method for the production of apharmaceutical composition comprising the steps of the method asdescribed supra; and the further step of formulating the compoundidentified and obtained or a derivative thereof in a pharmaceuticallyacceptable form.

The therapeutically useful compounds identified according to the methodsof the invention can be formulated and administered to a patient asdiscussed above. For uses and therapeutic doses determined to beappropriate by one skilled in the art and for definitions of the term“pharmaceutical composition” see infra.

Furthermore, the present invention encompasses a method for thepreparation of a pharmaceutical composition comprising the steps of theabove-described methods; and formulating a drug or pro-drug in the formsuitable for therapeutic application and preventing or ameliorating thedisorder of the subject diagnosed in the method of the invention.

Drugs or pro-drugs after their in vivo administration are metabolized inorder to be eliminated either by excretion or by metabolism to one ormore active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm.24 (1996), 449-459). Thus, rather than using the actual compound orinhibitor identified and obtained in accordance with the methods of thepresent invention a corresponding formulation as a pro-drug can be usedwhich is converted into its active in the patient. Precautionarymeasures that may be taken for the application of pro-drugs and drugsare described in the literature; see, for review, Ozama, J. Toxicol.Sci. 21 (1996), 323-329).

In a preferred embodiment of the method of the present invention saiddrug or prodrug is a derivative of a medicament as defined hereinafter.

The present invention also relates to a method of diagnosing a disorderrelated to the presence of a molecular variant of the CYP2C8 gene orsusceptibility to such a disorder comprising determining the presence ofa polynucleotide or the gene of the invention in a sample from asubject.

In accordance with this embodiment of the present invention, the methodof testing the status of a disorder or susceptibility to such a disordercan be effected by using a polynucleotide gene or nucleic acid of theinvention, e.g., in the form of a Southern or Northern blot or in situanalysis. Said nucleic acid sequence may hybridize to a coding region ofeither of the genes or to a non-coding region, e.g. intron. In the casethat a complementary sequence is employed in the method of theinvention, said nucleic acid molecule can again be used in Northernblots. Additionally, said testing can be done in conjunction with anactual blocking, e.g., of the transcription of the gene and thus isexpected to have therapeutic relevance. Furthermore, a primer oroligonucleotide can also be used for hybridizing to one of the abovementioned CYP2C8 gene or corresponding mRNAs. The nucleic acids used forhybridization can, of course, be conveniently labeled by incorporatingor attaching, e.g., a radioactive or other marker. Such markers are wellknown in the art. The labeling of said nucleic acid molecules can beeffected by conventional methods.

Additionally, the presence or expression of variant CYP2C8 gene can bemonitored by using a primer pair that specifically hybridizes to eitherof the corresponding nucleic acid sequences and by carrying out a PCRreaction according to standard procedures. Specific hybridization of theabove mentioned probes or primers preferably occurs at stringenthybridization conditions. The term “stringent hybridization conditions”is well known in the art; see, for example, Sambrook et al., “MolecularCloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor,1989; “Nucleic Acid Hybridisation, A Practical Approach”, Hames andHiggins eds., IRL Press, Oxford, 1985. Furthermore, the mRNA, cRNA, cDNAor genomic DNA obtained from the subject may be sequenced to identifymutations which may be characteristic fingerprints of mutations in thepolynucleotide or the gene of the invention. The present inventionfurther comprises methods wherein such a fingerprint may be generated byRFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNAmay be amplified prior to analysis, the methods of which are well knownin the art. RNA fingerprints may be performed by, for example, digestingan RNA sample obtained from the subject with a suitable RNA-Enzyme, forexample RNase T₁, RNase T₂ or the like or a ribozyme and, for example,electrophoretically separating and detecting the RNA fragments asdescribed above. Further modifications of the above-mentioned embodimentof the invention can be easily devised by the person skilled in the art,without any undue experimentation from this disclosure; see, e.g., theexamples. An additional embodiment of the present invention relates to amethod wherein said determination is effected by employing an antibodyof the invention or fragment thereof. The antibody used in the method ofthe invention may be labeled with detectable tags such as a histidineflags or a biotin molecule.

The invention relates to a method of diagnosing a disorder related tothe presence of a molecular variant of a CYP2C8 gene or susceptibilityto such a disorder comprising determining the presence of a polypeptideor the antibody of the invention in a sample from a subject.

In a preferred embodiment of the above described method said disorder isa cancer or cardiovascular disease.

In a preferred embodiment of the present invention, the above describedmethod is comprising PCR, ligase chain reaction, restriction digestion,direct sequencing, nucleic acid amplification techniques, hybridizationtechniques or immunoassays. Said techniques are very well known in theart.

Moreover, the invention relates to a method of detection of thepolynucleotide or the gene of the invention in a sample comprising thesteps of

-   (a) contacting the solid support described supra with the sample    under conditions allowing interaction of the polynucleotide or the    gene of the invention with the immobilized targets on a solid    support and;-   (b) determining the binding of said polynucleotide or said gene to    said immobilized targets on a solid support.

The invention also relates to an in vitro method for diagnosing adisease comprising the steps of the method described supra, whereinbinding of said polynucleotide or gene to said immobilized targets onsaid solid support is indicative for the presence or the absence of saiddisease or a prevalence for said disease.

The invention furthermore relates to a diagnostic composition comprisingthe polynucleotide, the gene, the vector, the polypeptide or theantibody of the invention.

In addition, the invention relates to a pharmaceutical compositioncomprising the polynucleotide, the gene, the vector, the polypeptide orthe antibody of the invention. These pharmaceutical compositionscomprising, e.g., the antibody may conveniently be administered by anyof the routes conventionally used for drug administration, for instance,orally, topically, parenterally or by inhalation. Acceptable saltscomprise acetate, methylester, HCl, sulfate, chloride and the like. Thecompounds may be administered in conventional dosage forms prepared bycombining the drugs with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable character or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.The carrier(s) must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not deleterious to therecipient thereof. The pharmaceutical carrier employed may be, forexample, either a solid or liquid. Exemplary of solid carriers arelactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax.

The dosage regimen will be determined by the attending physician andother clinical factors; preferably in accordance with any one of theabove described methods. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment.

Furthermore, the use of pharmaceutical compositions which compriseantisense-oligonucleotides which specifically hybridize to RNA encodingmutated versions of the polynucleotide or gene according to theinvention or which comprise antibodies specifically recognizing amutated polypeptide of the invention but not or not substantially thefunctional wild-type form is conceivable in cases in which theconcentration of the mutated form in the cells should be reduced.

In another embodiment the present invention relates to the use of thepolynucleotide, the gene, the vector, the polypeptide, thepolynucleotides having at a position corresponding to position 270 (exon3) and 206 (exon 8) (GenBank accession No: AF136833.1 and AF136842.1,respectively) of the CYP2C8 gene (GenBank accession No: GI: 13787189) anA instead of a G in position 270 and a G instead of an A in position206, or the antibody of the invention for the preparation of adiagnostic composition for diagnosing a disease.

Thanks to the present invention the particular drug selection, dosageregimen and corresponding patients to be treated can be determined inaccordance with the present invention. The dosing recommendations willbe indicated in product labeling by allowing the prescriber toanticipate dose adjustments depending on the considered patient group,with information that avoids prescribing the wrong drug to the wrongpatients or at the wrong dose.

In a further embodiment the present invention relates to the use of thepolynucleotide, the gene, the vector, the polypeptide, thepolynucleotides having at a position corresponding to position 117 inexon 5 (GenBank accession No: AF136837.1) of the CYP2C8 gene (GenBankaccession No: GI: 13787189) a T instead of an A, or the antibody of theinvention for the preparation of a pharmaceutical composition fortreating a disease.

In a more preferred embodiment of the use of the present invention saiddisease is an incompatibility or disease related to arachidonic acidmetabolism, cancer or cardiovascular disease.

Finally, the present invention relates to a diagnostic kit for detectionof a single nucleotide polymorphism comprising the polynucleotide, thegene, the vector, the polypeptide, the antibody, the host cell, thetransgenic non-human animal or the solid support of the invention.

The kit of the invention may contain further ingredients such asselection markers and components for selective media suitable for thegeneration of transgenic cells and animals. The kit of the invention canbe used for carrying out a method of the invention and could be, interalia, employed in a variety of applications, e.g., in the diagnosticfield or as research tool. The parts of the kit of the invention can bepackaged individually in vials or other appropriate means depending onthe respective ingredient or in combination in suitable containers ormulticontainer units. Manufacture of the kit follows preferably standardprocedures which are known to the person skilled in the art. The kit maybe used for methods for detecting expression of a mutant form of thepolypeptides, genes or polynucleotides in accordance with any one of theabove-described methods of the invention, employing, for example,immunoassay techniques such as radio-immunoassay or enzyme-immunoassayor preferably nucleic acid hybridization and/or amplification techniquessuch as those described herein before and in the Examples as well aspharmacokinetic studies when using non-human transgenic animals of theinvention.

The invention will now be described by reference to the followingbiological Examples which are merely illustrative and are notconstructed as a limitation of the scope of the present invention.

EXAMPLES Example 1 Isolation of Genomic DNA from Human Blood, Generationand Purification of CYP2C8 Gene Fragments

Genomic DNA was obtained by standard ion exchange chromatographytechniques (Qiagen kits for isolation of genomic DNA from blood). Bloodfrom all the individuals tested (volunteers from Parexel, Berlin and theInstitute for Clinical Pharmacology, Stuttgart) was obtained underconsideration of all legal, medical and bureaucratical requirements.Further samples from other ethnic populations (e.g. Caucasian, Japanese,African-American) were purchased from commercial sources.

By using polymerase chain reaction (PCR) with specific oligonucleotideprimers, two for each fragment, defined DNA-fragments containingspecific parts of the human CYP2C8 gene were obtained. These specificoligonucleotide primers were designed to bind to sequences upstream anddownstream of the various exons as well as in the 5 prime region of theCYP2C8 gene. The resulting DNA fragments did not contain codogenic partsalone but also sequences covering the intronic parts located at theexon-intron boundaries. These sites are known to be important forcorrect processing and subsequent expression of the protein encodingmRNA, a process called “splicing”. Commercially synthesizedoligonucleotide primer pairs that were purified by affinitychromatography were optimized for each of the 9 exon and 4 promoterfragments of the human CYP2C8 gene. The sequences for each primer arelisted in table 1.

Polymerase chain reactions were performed under conditions that wereoptimized for each of the nine fragments and a promoter region coveringabout 2 kb upstream of the initiation codon for mRNA translation. PCRswere carried out for all exons in a volume of 50 μl. 10-50 ng oftemplate DNA were added to standard PCR-buffer containing 1.5 mM MgCl₂,200 μM dNTPs and 1 U Taq-polymerase (all from Qiagen, Hilden) as well as10-40 pMol of primers (MWG Biotech, Munich). All PCR reactions wereperformed on identical conditions at a Perkin Elmer thermocycler (Modell9700) with an initial denaturation step of 94° C. for 2 min, followed by34 cycles for PCR-fragment generation with 45 s denaturation at 94° C.,45 s of annealing at 62° C. and 1 min at 72° C. for elongation. Theexact location of the primers and size of the desired fragments are alsolisted in table 1.

The defined DNA fragments containing specific parts of the CYP2C8 gene,exon as well as some intron sequences at the inton-exon boundaries wereprocessed to remove nonincorporated nucleotides and buffer componentsthat might otherwise interfere with the subsequent determination of theindividual CYP2C8 genotype by direct cycle sequencing. For thispurification, standard ion exchange chromatography techniques were used(Qiagen kits for PCR-fragment purification). For all fragmentssufficient yields of purified fragments, suitable for direct DNAsequencing analysis were obtained. Aliquots of purified fragments weresubjected to direct sequence analysis of the CYP2C8 gene in an ABI 3700capillary sequencer.

Example 2 Identification of Different CYP2C8 Alleles by SequenceDetermination in Various Individuals

For sequence analysis of relevant regions of the human CYP2C8 gene frommany different individuals, PCR amplifications of the relevant regionsof the gene were carried out (pimers see table 1) following purificationof the PCR products and sequencing with established methods (ABI dyeterminator cycle sequencing). Since the individual genetic makeup isrepresented by two copies of any gene (diploidy), great care has to betaken in the evaluation of the sequences not to unambiguously identifyhomozygous, but also heterozygous sequence variation. Therefore, incases where no clear discrimination could be detected, forward andreverse sequencing was performed. Moreover, for the discovery ofcomplete and defined alleles, e.g. in linkage equilibrium, it isnecessary to cover all exons as well as the promoter region to provide acomprehensive basis for the phenotype prediction of individual SNPs.

For the evaluation of CYP2C8 variations in the human population,sequence analyses of the relevant regions, including a 2 kb pormoterfragment and all exons of the gene were carried out from genomic DNA ofeach 48 Caucasian, Japanese and African-American individuals. Thesequences were subjected to a computer analysis programme (Phredphrap™,Perkin Elmer) and inspected manually for the occurrence of DNA sequencesdeviating from recently published CYP2C8 sequences that were consideredto represent the “wild type” sequences in this work.

Because population genetics enables a calculation of the expectedfrequency of homozygous vs. heterozygous alleles of a defined gene(Hardy Weinberg formula: 2p e2+2pq+2q e2=1), it was possible to confirmpredicted distributions of homozygous vs. heterozygous alleles anddeviations from the experimental findings. This serves as experimentalcontrol that a detected sequence variation indeed represents a novelallele.

Several new CYP2C8 sequence variations were discovered andexperimentally confirmed using this approach which are shown in table 2.18 polymorphisms are located in the 5′ untranslated region/promoter ofthe gene (GenBank accession No: AF136830.1). 22 new polymorphisms couldbe found in sequences of introns 1, 2, 3, 4 and 8 (GenBank accessionNos: AF136832.1, AF136833.1, AF136835.1 AF136843.1 and AF136844.1) andone in the 3′ untranslated region of the gene (GenBank accession No:AF136845.1).

Furthermore, three particular nucleotide changes in exon 3 (position 334at exon/intron boundary, GenBank accession No: 163833.1) and exon 8(position 30 and 87, GenBank accession No: AF1368.43.1) were detectedthat do not change the amino acid sequence. In exon 3 (position 329,GenBank accession No: AF136833.1), exon 4 (position 243, GenBankaccession No: AF136842.2 and position 13 of GenBank accession No:AF136835.1), exon 5 (position 42, 101 and 104, GenBank accession No:AF136837.1), exon 6 (position 309, GenBank accession No: AF136838.1) and7 (position 1135, GenBank accession No: NM_(—)000770.1 and position 232,GenBank accession No: AF136840.1) nine SNPs could be identified thatchange the protein sequence as shown in table 4, where the deviativeamino acid is typed in a bold style. These novel, and already publishedCYP2C8 SNPs serve as markers for the characterization of the CYP2C8status in patients.

The positions of the novel CYP2C8 SNPs, including the exact novelsequence context are listed in table 2. The deviative base in thesequence is typed underlined and in a bold style.

Example 3 Determination of the CYP2C8 Promoter Allele ContainingG-1207A, delAT −640 to −641 as a Pharmacogenetic Factor Influencing DrugLevels

The anticancer drug TAXOL (paclitaxel) can be considered to be theprototype for CYP2C8 and it's isoform 6-hydroxypaclitaxel as diagnosticsubstrate. Furthermore, verapamil represents another specific substratefor CYP2C8 that is methylated to different metabolites, e.g D-702 andD-703 or desalkylated to D-617 (i.e. a substrate of CYP3A4). Thegeneration of a specific metabolite used to selectively proof thefunctional activity of CYP2C8 (LC-MS) is shown in FIG. 4. In parallel,the amount of enzyme is determined by western blotting (see example 9).

Using these analytical tools, phenotypically characterized samples thathave been treated with TAXOL (paclitaxel) (N=22) or verapamil (N=15, 44)were subjected to genotyping. Either of the collectives showed that twoSNPs G-1207A, delAT-640 to −641 of the 5′ untranslated region are inlinkage disequilibrium and, in combination with SNPs G270A (exon 3) andA206G (exon 8), represent a new allele (haplotype) that is mainlydefined by the presence of the two novel promoter SNPs G-1207A anddelAT-640 to −641. FIG. 4 shows that this allele is responsible for thereduced level of metabolized substrate.

Example 4 Functional Consequences of the Identification of CYP2C8Promoter Polymorphisms

The eukaryotic promoter region of a gene is composed of severalregulatory elements, e.g enhancer, silencer and other responsiveelements. Here, single nucleotide polymorphisms exhibit significantinfluence. Regarding cytochrome P450-enzymes induction mechanisms, e.g.transcription factors like C/EBP, HPFs or barbiebox-sites identified inCYP2C9 (Klose, J Biochem Mol Toxicol 13 (1999), 289-95) are importantsince temporary expression is required. SNPs change or interfere withsuch elements and can alter promoter action and/or transcriptionactivity. The novel SNP identified at position −1207 most probablyabolishes transcription factor binding that has selectively been shownfor the binding of tissue specific sterol regulatory element bindingprotein 1 (SREBP-1, Valleft, J Biol. Chem. 271 (1996), 12247-53).Reduced expression has been shown if position −1207 does not correspondto the wild type. The correlation of this new allele with CYP2C8 enzymeactivity is displayed in FIG. 4. The linkage with another unidentifiedSNPs further downstream in the promoter region of CYP2C8 (delAT −640 to−641) confers new value to the allele in respect to diagnosticapplications.

Example 5 Determination of the CYP2C8 Promoter Polymorphism T-370G as aPharmacogenetic Factor Influencing Drug Levels

Another polymorphism located further downstream at position 1668 in the5-prime untranslated region (position −370 relative to the ATG-startcodon) could be identified to significantly increase the expressionlevel as shown in FIG. 3. This allele refers to as an extensivemetabolizing phenotype (EM) that was confirmed by investigation ofphenotypically characterized samples. In several cases, singlenucleotide polymorphisms G-1207A, delAT −640 to −641 occur incombination with T-370G. LC-MS results from these samples show thatindividuals carrying the T-370G alone have an increased CYP2C8-activityas compared to those heterozygous for the polymorphisms G-1207A, delAT−640 to −641 allele. Concerning the application in a diagnostic assaythese data clearly show the influence of position T-370G on theexpression levels of CYP2C8, i.e. the latter SNP is responsible for achange from poor (PM) to intermediate/extensive metabolism (IM/EM) asdemonstrated in example 9/table 6.

Independently, a further validation was carried out by genotyping 22samples corresponding to individual liver extracts, in which themetabolism of TAXOL (paclitaxel) was assessed. The data confirm resultsas presented in examples 8 and 9. FIG. 2 shows the significantcorrelation between a heterogeneous polymorphism at position −370G andthe increase of CYP2C8 protein levels. The liver extract from a samplehomogeneous for position −370 displayed the highest CYP2C8-protein level(>400 pmol/mg protein). This is an additional independent result thatsupports the significant correlation in FIG. 2.

Example 6 Determination of the CYP2C8 Polymorphism at Amino AcidPosition 264 (Exon 5) as a Pharmacogenetic Factor Influencing DrugLevels

In another embodiment the present invention relates to a polymorphism inexon 5 at position C104G (GenBank accession No: AF136837.1). This changecorrelates with a reduced protein concentration analyzed from genotypedsamples (FIG. 1) that could be due to less stable mRNA or protein. Thepolypeptides encoded by the polynucleotides of the invention may havealtered biological or immunological properties due to the polymorphismsreferred to in accordance with the present invention. Examples for saidaltered properties are stability of the polypeptides which may beeffected or the incapability to effectively metabolize certain drugs.

Example 7 Using Restriction Fragment Length Polymorphism (RFLP) Analysisto Detect SNPs Relevant in Phenotypic Prediction

CYP2C8-polymorphisms can be detected not only by sequencing but also byvarious other means. As one alternative to the sequencing methodology,genotyping can be performed with PCR fragments to be processed by onerestriction endonuclease specifically cutting at a region, composed of aunique sequence of 4-6 nucleotides. Due to the limited length of aPCR-fragment sometimes advantage can be taken of this specificity if itdiscriminates mutant and wild type, i.e. resulting in digested or undigested fragments (Table 5). Regarding the present invention this wasthe case for fragments of the promoter region 4 (position −370, doublecutter AcsI for wild type and single cutter in the mutant), thatindicates the respective allele, exon 3 (position 275, single cutterSapI for CYP2C8-mutant), and exon 5 (position 104, single cutter ClaIfor wild type). Depending of the fragments' specific SNP-region therestriction pattern unambiguously reflects either the wild type, theheterozygous or homozygous mutant. As defined by the primers listed intable 1, the exons screened for result in the following RFLP-fragments:

TABLE 5 Length SNP position (bp) Genotype (PCR-fragment) Enzyme uncutwt/wt (bp) wt/mut (bp) mut/mut (bp) # −370, (5′UTR) Acs1 483 33/150/30033/150/183/300 183/300 # 270, (exon 3) Sapl 328 328 149/179/328 149/179# 104, (exon 5) Clal 584 192/392 192/392/584 584

Example 7 Identification of New CYP2C8 Polymorphisms by SequenceAnalysis of a Collection of Various Individuals from Different EthnicGroups

The screen for SNPs in the CYP2C8 gene in the genomes of differentethnic groups yielded a number of polymorphisms listed in table 2. 48samples were analyzed from each of the ethnic populations Caucasian,Japanese and African-American, respectively. Within this collection, thelarge number of SNPs in the untranslated region could be considered topotentially influence the protein level.

Furthermore, several polymorphisms show extensive inter ethnicaldiscrepancies (Table 3) between various ethnical groups. The 57 newpolymorphisms identified in the CYP2C8 gene will complement the existingknowledge and contribute to a more comprehensive understanding of thegene, avoiding problems in drug response and, concerning other ethnicalgroups, thus facilitating “bridging studies” that could be of interestwhen projecting data from these embodiment.

Example 8 Characterization of Promoter SNPs by Transfection ofPromoter-Reporter Plasmids into Human Cells

Three promoter SNPs were tested for their contribution to differentexpression levels by transfection assays using the LS174T cell line.Promoter fragments containing the wild type or the SNPs at positionscorresponding to positions G-1207A and delAT −640 to −641 (GenBankaccession No: AF136830.1) or at position corresponding to positionT-370G (GenBank accession No: AF136830.1) were introduced into acommercial mammalian expression vector. The plasmid harbours standardsequences for the propagation in eukaryotic cells including the reportergene luciferase that is controlled by the integrated promoter sequence.Following sequence verification of each DNA-insert, cells werecotransfected with β-galactosidase, harvested after 48 h and analysedfor luciferase activity. Promoter activities (%) are shown in FIG. 5following normalization to the transfection efficacy as determined byβ-galactosidase detection. The data are in full agreement with theobservations from phenotypically characterized samples. A DNA-constructthat contains the 1207G>A and −640 to −641 delAT polymorphisms (FIG. 5)showed decreased transcription for the CYP2C8-promoter levels comparedto the wild type. In contast, the reporter plasmid revealed increasinglevels of luciferase protein under control of a CYP2C8-promotercontaining a polymorphism corresponding to position −370.

Example 9 Protein Quantification of Samples Containing SNPs at PromoterPosition G-1207A, delAT −640 to −641, T-370G and C104G (exon 5, aminoacid Position 264) of CYP2C8

Protein extracts have been prepared from human liver samples. Theprotein levels of CYP2C8 were analysed by western blot using samplesgenotyped for SNPs G-1207A, delAT −640 to −641, T-370G (all GenBankaccession No: AF136830.1) and C104G (GenBank accession No: AF136837.1).Table 5 shows the effects of different genotypes on the expressionlevels of CYP2C8 (pmol/mg) normalized for the wild type (=100%). Resultsare in total agreement with the functional data described by thepromoter-reporter assays in example 8. The promoter SNP in positionT-370G confers to increased levels of the CYP2C8 protein (150%), whereaspolymorphisms G-1207A and delAT −640 to −641 in contrast show a reducedprotein expression (72%). In combination with SNPs G-1207A and delAT−640 to −641, or C104G alone the polymorphism T-370G differentiallyinfluences protein levels as indicated by arrow. Here, the presence oftwo SNPs with significant frequency leads to combined effects.Therefore, considerations for reliable phenotype prediction as a resultfrom genotyping must depend on multiple SNP-analyses. The data indicatethat the SNP in position −370, which by itself is responsible for upregulation of the CYP2C8-protein level, shows no significant CYP2C8increase if it is combined with SNPs G-1207A and delAT −640 to −641(5′UTR), which by themselves reduce the expression. The combinedexpression level of 119% is barely higher than in the homozygouswildtype situation. Vice versa, in combination with the C104G allele(exon 5) the increased expression due to the SNP at position −370 iscompensated by the SNP C104G to normal expression levels (97%) comparedto the wild type. This reflects the strong impact of SNP C104G alone onthe protein level (see FIG. 1). The SNP C104G therefore represents anallele for down regulation.

TABLE 6 Genotype (by detection of listed SNPs) CYP 2C8-levels (%)Effects Wild type 100 No G-1207A, delAT −640 72 ↓ to −641 (5′UTR) T-370G(5′UTR) 150 ↑ G-1207A, delAT −640 119 ↑↓ to −641 and T-370G (5′UTR)T-370G (5′UTR) and 97 ↑↓ aa I264M (exon 5) No sample with aa changeI264M or G-1207A, delAT −640 to −641 (5′UTR) and I264M was detected.

Example 10 Pharmacogenetic Relevance of the CYP2C8 Polymorphisms atPosition 329 (exon 3, Thr 159 Pro), 309 (exon 6, Glu 274 stop) and 1135(exon 7, Gly 365 Ser)

In another embodiment the present invention relates to a polymorphismsin exon 3, exon 6 and 7 at positions 329 (GenBank accession No:AF136833.1), 309 (GenBank accession No: AF136838.1) rand 1135 (GenBankaccession No: NM_(—)000770.1) respectively. The delA change in position329 causes a frameshift abolishing the C-terminal part of the protein.The G309T change in position 309 results in a premature termination atamino acid position 274 of the protein. Both variant transcripts encodefor polypeptides that will loose their function and are therefore mostlikely poor metabolizer alleles (FIG. 6). In another embodiment thepresent invention relates to a polymorphism at position G1135A in exon 7(GenBank accession No: NM_(—)000770.1). This substitution results in achange from Glycin to Serin at position 365 within the active site ofthe CYP2C8 enzyme (FIG. 6). Because the active site of wildtype CYP2C8contains hydrophobic amino acids to enable the hydrohpilic substrate toefficiently enter the substrate pocket, this amino acid exchange to ahydrophilic residue will severely interfere with substrate binding andsubsequent metabolism.

Table 1 Primer sequences for the generation of CYP2C8 PCR-fragments (SEQID NOS: 25-51, respectively, in order of appearance)

TABLE 1 Primer sequences for the generation of CYP2C8 PCR-fragments (SEQID NOS: 25-51, respectively, in order of appearance) All primerlocations refer to different contigs of HTGS-Database, GenBank Acc. No.AL359672.10 PCR-fragments at the 5′UTR are overlapping. PCR- PCR-fragment fragm. Contig spec. name size (bp) exon location Primerposition Primer Sequence (5′ - 3′) Contig 115244-120972 (5629 bp) 5′UTRFragm. 1 537 534 120439-120416 forward: ATT TTA GTC AAT CTT GGT GGC CCG119902-119926 reverse: TTC AAC AGA AGA TGG AAC ACA GGG A 5′UTR Fragm. 2545 120004-119980 forward: TCA TGA CCA TTG ACT ATC AGT TCC C119460-119483 reverse: TGA TAC CCA TTG GGG TTC ATT ACC 5′UTR Fragm. 3751 119576-119552 forward: AAC AGA GTC AAG GTG GCG TAT CTT C118826-118854 reverse: CAA TAT TCT CAG ATT AAT GAC CAG TTG GG Sequencing118938-118963 AGA CTT AGC CCT TGA TAA CAA AAG CC primer 5′UTR Fragm. 4483 −2486 119008-118982 forward: GTT TAG GCA GCT GTA TTT TAA GTG AAC118526-118550 reverse: ACT CCA AAG TTT TTA TAA CAC TCC C Exon 1 4722487-2654 118687-118664 forward: GGC ACT GGA AAG AAG GAG TAG GAC118216-118242 reverse: GAT CTA TTA TAA TAG TGT GCT TCC AGG Exon 2 4574198-4360 116999-116978 forward: TTG TGT ACC AAT TGC CTG GGT C116543-116566 reverse: TTT TTA GGG CTC TGT TTT CCA TCC Exon 3 3284532-4681 116531-116508 forward: GAG CTT AGC CTA TCT GCA TGG CTG116204-116223 reverse: ACC TGG CCA CCC CTG AAA TG Contig 78619-85206(6588 bp) Exon 4 541 1378-1538 83947-83970 forward: TCC ATG CTG ATT TTTTTT GGA CAC Alternative AF136834.2 44-67 83429-83450 reverse: CTG ACCCCT TGC ACT TCT GAT G Contig 138518-143654 (5137 bp) Exon 5 5831319-1495 139593-139617 forward: TGA CGA GTT ATT GGG TGC AGT ACA C140176-140154 reverse: TTC CAT GAT GTT TAG TGC AGG CC Contig85307-115243 (29937 bp) Exon 6 519 8875-9016 93884-93903 forward: TTGAAG TAA GAC AGG GCA TCG G 94402-93379 reverse: AGA AAC AAG GTG GAG GATACT GGC Exon 7 328 11749-11936 96986-97009 forward: GGC CAT GAA TTG CTATGA CAA ATG 97313-97290 reverse: GGT TGG AAC CAA ACC AGC ACT ATG Exon 8462 15788-15929 100976-100997 forward: CTG GCT GGA CCT GAG TTT CCT C101437-101418 reverse: TTA ACT CCT GCA AGC CCC GC Exon 9/3′UTR 54317526-17707 102630-102652 forward: GTA CAT TTG TTT GTC CCA CCA TCC103172-103149 reverse: TGC AGT GAC CTG AAC AAC TCT CCT

Table 2 SNPs identified in the CYP2C8 gene (SEQ ID NOS: 52-213, 382-387,214-303, 388-393, 304-309, 394-399 and 310-375, respectively, in orderof appearance)

TABLE 2SNPs identified in the CYP2C8 gene (SEQ ID NOS: 52-213, 382-387, 214-303,388-393, 304-309, 394-399 and 310-375, respectively, in order of appearancevariant   PCR- position fragm GenBank (relative wild type/mutantmutant/mutant Location Acc. No to ATG) wild type (f) and (r) (f) and (r)  (f) and (r) 5'UTR- AF136830.1 #306 f:GATGTGATG AGTGTGAAAAT f:GATGTGATG( AG )TGTGAAAAT f:GATGTGATGTGTGAAAAT fragm 1 to 307r:ATTTTCACACTCATCACATC r:ATTTTCACA( CT )CATCACATC r:ATTTTCACACATCACATC(= -1731 to -1732) 5'UTR- AF136830.1 #411 f:GGAAATAAC T GTACTGGTCf:GGAAATAAC T/A GTACTGGTC f:GGAAATAAC A GTACTGGTC fragm 1 = -1627)r:GACCAGTAC A GTTATTTCC r:GACCAGTAC A/T GTTATTTCC r:GACCAGTAC TGTTATTTCC 5'UTR- AF136830.1 #560 f:GGTCTGCAC A TTGCAGTGG f:GGTCTGCAC A/GTTGCAGTGG f:GGTCTGCAC G TTGCAGTGG fragm 1 (= -1478) r:CCACTGCAA TGTGCAGACC r:CCACTGCAA C/T GTGCAGACC  r:CCACTGCAA C GTGCAGACC 5'UTR-AF136830.1 #713 f:AAAACAATA G AAGCAGCCA f:AAAACAATA G/T AAGCAGCCAf:AAAACAATA T AAGCAGCCA fragm 2 (-1325) r:TGGCTGCTT C TATTGTTTTr:TGGCTGCTT A/C TATTGTTTT r:TGGCTGCTT A TATTGTTTT 5' UTR- AF136830.1#817 f:AGTGCTGAA C AACTTTCAC f:AGTGCTGAA C/A AACTTTCAC  f:AGTGCTGAA AAACTTTCAC fragm 2 (= -1221) r:GTGAAAGTT G TTCAGCACT  r:GTGAAAGTT T/GTTCAGCACT  r:GTGAAAGTT T TTCAGCACT 5'UTR- AF136830.1 #824 f:AACAACTTT CACTTGTGAG f:AACAACTTT C/A ACTTGTGAG f:AACAACTTT A ACTTGTGAG fragm 2 (=-1214) r:CTCACAAGT G AAAGTTGTT r:CTCACAAGT T/G AAAGTTGTT r:CTCACAAGT TAAAGTTGTT 5'UTR- AF136830.1 #831 f:TTCACTTGT G AGGTGATGC f:TTCACTTGT G/AAGGTGATGC f:TTCACTTGT A AGGTGATGC fragm 2 (= -1207) r:GCATCACCT CACAAGTGAA r:GCATCACCTT/CACAAGTGAA r:GCATCACCT T ACAAGTGAA 5'UTR-AF136830.1 #879 f:CTTTTGAGC G TCTCCGGTC f:CTTTTGAGC G/A TCTCCGGTC f:CTTTTGAGC A TCTCCGGTC fragm 2 (= -1159) r:GACCGGAGA C GCTCAAAAGr:GACCGGAGA T/C GCTCAAAAG r:GACCGGAGA T GCTCAAAAG 5'UTR- AF136830.1 886 f:GCGTCTCCG G TCCTCTTAT f:GCGTCTCCG G/T TCCTCTTAT f:GCGTCTCCG TTCCTCTTAT fragm 2 (= -1152) r:ATAAGAGGA C CGGAGACGC r:ATAAGAGGA A/CCGGAGACGC r:ATAAGAGGA A CGGAGACGC 5'UTR- AF136830.1 #1058 f:ACCCCAATG GGTATCAGAA f:ACCCCAATG G/A GTATCAGAA f:ACCCCAATG A GTATCAGAA fragm 3 =-980) r:TTCTGATAC C CATTGGGGT r:TTCTGATAC T/C CATTGGGGT r:TTCTGATAC TCATTGGGGT 5'UTR- AF136830.1 #1271 to f:GTATTTATG TTA TTATTATGTf:GTATTTATG( TTA) f:GTATTTATGTTATTATGT fragm 3 1273  TTATTATGT (= -765)r:ACATAATAA TAA CATAAATAC r:ACATAATAA( TAA) r:ACATAATAACATAAATACto (-767) CATAAATAC 5'UTR- AF136830.1 #1397 to f:TGTAATAAC AT ATATATTTAf:TGTAATAAC (AT) ATATATTTA f:TGTAATAACATATATTTA fragm 3 1398 r:TAAATATATAT GTTATTACA r:TAAATATAT (AT) GTTATTACA r:TAAATATATGTTATTACA (= -640)to (-641) 5'UTR- AF136830.1 #1627 f:TTTTTTATA T ACAAAATAT f:TTTTTTATAT/C ACAAAATAT f:TTTPTTATA C ACAAAATAT fragm 4 (= -411) r:ATATTTTGT ATATAAAAAA r:ATATTTTGT G/A TATAAAAAA r:ATATTTTGT G TATAAAAAA 5'UTR-AF136830.1 #1668 f:GGTCATAAA T TCCCAACTG f:GGTCATAAA T/G TCCCAACTGf:GGTCATAAA G TCCCAACTG fragm 4 (= -370) r:CAGTTGGGA A TTTATGACCr:CAGTTGGGA C/A TTTATGACC r:CAGTTGGGA C TTTATGACC 5'UTR- AF136830.1#1767 f:ACATTGGAA C AACCAGGGA f:ACATTGGAA C/A AACCAGGGA f:ACATTGGAA AAACCAGGGA fragm 4 = -271) r:TCCCTGGTT G TTCCAATGT r:TCCCTGGTT T/GTTCCAATGT r:TCCCTGGTT T TTCCAATGT 5'UTR- AF136830.1 #1785/ f:AATTAAAAATACCTGGGC  f:AATTAAAAA (A) TACCTGGGC f:AATTAAAAA A TACCTGGGCfragm 4 1786 r:GCCCAGGTATTTTTAATT r:GCCCAGGTA (T) TTTTTAATT r:GCCCAGGTAT TTTTTAATT (= -252) 5'UTR- AF136830.1 #1887 f:CTATCCATG G GCCAAAGTCf:CTATCCATG G/A GCCAAAGTC f:CTATCCATG A GCCAAAGTC fragm 4 (= -151)r:GACTTTGGC C CATGGATAG r:GACTTTGGC T/C CATGGATAG r:GACTTTGGC TCATGGATAG 5'UTR- AF136830.1 #1905 f:CCACTCAGA A AAAAAGTAT f:CCACTCAGAA/C AAAAAGTAT f:CCACTCAGA C AAAAAGTAT fragm 4 (= -133) r:ATACTTTTT TTCTGAGTGG r:ATACTTTTT G/T TCTGAGTGG r:ATACTTTTT G TCTGAGTGG 5'UTR-AF136830.1 #1952 f:ACATGTCAA A GAGACACAC f:ACATGTCAA A/C GAGACACACf:ACATGTCAA C GAGACACAC fragm 4 (= -86) r:GTGTGTCTC T TTGACATGTr:GTGTGTCTC G/T TTGACATGT r:GTGTGTCTC G TTGACATGT Intron 1 AF136832.1#171 f:ATTCAGAAA T ATCGAATCT f:ATTCAGAAA T/C ATCGAATCT f:ATTCAGAAA CATCGAATCT r:AGATTCGAT A TTTCTGAAT r:AGATTCGAT G/A TTTCTGAAT r:AGATTCGATG TTTCTGAAT Intron 1 AF136832.1 #258 f:AGCAAATAG C GACTTATTT f:AGCAAATAGC/T GACTTATTT f:AGCAAATAG T GACTTATTT r:AAATAAGTC G CTATTTGCTr:AAATAAGTC A/G CTATTTGCT r:AAATAAGTC A CTATTTGCT Intron 2 AF136833.1#122 f:ATGGCTGCC G AGTGTTGCA f:ATGGCTGCC G/A AGTGTTGCA f:ATGGCTGCC AAGTGTTGCA r:TGCAACACT C GGCAGCCAT r:TGCAACACT T/C GGCAGCCAT r:TGCAACACTT GGCAGCCAT Intron 2 AF136833.1 #150 f:TCCTTGGCT G TGAATTCTC f:TCCTTGGCTG/A TGAATTCTC f:TCCTTGGCT A TGAATTCTC r:GAGAATTCA C AGCCAAGGAr:GAGAATTCA T/C AGCCAAGGA r:GAGAATTCA T AGCCAAGGA Intron 2 AF136833.1#180/181 f:CCTTTTTTTATTAGGAAT f:CCTTTTTTT (T) ATTAGGAAT f:CCTTTTTTT TATTAGGAAT r:ATTCCTAATAAAAAAAGG r:ATTCCTAAT (A) AAAAAAAGG r:ATTCCTAAT AAAAAAAAGG Intron 2 AF136833.1 #182 f:CTTTTTTTA T TAGGAATCAf:CTTTTTTTAT/CTAGGAATCA f:CTTTTTTTA C TAGGAATCA r:TGATTCCTA A TAAAAAAAGr:TGATTCCTA G/A TAAAAAAAG r:TGATTCCTA G TAAAAAAAG Exon 3 AF136833.1 #270f:TGGGGAAGA G GAGCATTGA f:TCGGGAAGA G/A GAGCATTGA f:TGGGGAAGA AGAGCATTGA r:TCAATGCTC C TCTTCCCCA r:TCAATGCTC T/C TCTTCCCCA r:TCAATGCTCT TCTTCCCCA Exon 3 AF136833.1 #334 f:AAAAACCAA G GGTGGGTGA f:AAAAACCAAG/A GGTGGGTGA f:AAAAACCAA A GGTGGGTGA r:TCACCCACC C TTGGTTTTTr:TCACCCACC T/C TTGGTTTTT r:TCACCCACC T TTGGTTTTT Exon 3 AF136833.1 #329f:TTGAGAAAA A CCAAGGGTG f:TTGAGAAAA( A) CCAAGGGTG f:TTGAGAAAACCAAGGGTGr:CACCCTTGGTTTTTCTCAA r:CACCCTTGG( T) TTTTCTCAA r:CACCCTTGGTTTTCTCAAIntron 3 AF136833.1 #378 f:CAGTTACCT G TCTTCACTA f:CAGTTACCT G/CTCTTCACTA f:CAGTTACCT C TCTTCACTA r:TAGTGAAGA C AGGTAACTG  r:TAGTGAAGAG/C AGGTAACTG r:TAGTGAAGA G AGGTAACTG Intron 3 AF136834.2 #87f:TGTAAGATA T GTTTAAAAT f:TGTAAGATA (T) GTTTAAAAT f:TGTAAGATAGTTTAAAATr:ATTTTAAAC A TATCTTACA r:ATTTTAAAC (A) TATCTTACA r:ATTTTAAACTATCTTACAIntron 3 AF136834.2 #162 f:ATAATTTTT T TAAAAATTT f:ATAATTTTT T/ATAAAAATTT f:ATAATTTTT A TAAAAATTT r:AAATTTTTA A AAAAATTAT r:AAATTTTTAT/A AAAAATTAT r:AAATTTTTA T AAAAATTAT Intron 3 AF136834.2 #163f:TAATTTTTT T AAAAATTTT f:TAATTTTTT T/A AAAAATTTT f:TAATTTTTT AAAAAATTTT r:AAAATTTTT A AAAAAATTA  r:AAAATTTTT T/A AAAAAATTA r:AAAATTTTTT AAAAAATTA Exon 4 AF136834.2 #243 f:ATCTGCTCC G TTGTTTTCC f:ATCTGCTCCG/A TTGTTTTCC f:ATCTGCTCC A TTGTTTTCC NM_000770.1 #583 r:GGAAAACAA CGGAGCAGAT r:GGAAAACAA T/C GGAGCAGAT r:GGAAAACAA T GGAGCAGAT Exon 4AF136835.1 #13 f:GGATTCTGA A CTCCCCATG f:GGATTCTGA A/G CTCCCCATGf:GGATTCTGA G CTCCCCATG r:CATGGGGAG T TCAGAATCC r:CATGGGGAG C/TTCAGAATCC  r:CATGGGGAG C TCAGAATCC Intron 4 AF136835.1 #180 f:TGATTTCCTG TTCAAAATT f:TGATTTCCT G/A TTCAAAATT f:TGATTTCCT A TTCAAAATTr:AATTTTGAA C AGGAAATCA r:AATTTTGAA T/C AGGAAATCA r:AATTTTGAA TAGGAAATCA Intron 4 AF136836.1 #116 f:ACTTAAAGT A TAATAAAAA f:ACTTAAAGTA/G TAATAAAAA f:ACTTAAAGT G TAATAAAAA r:TTTTTATTA T ACTTTAAGTr:TTTTTATTA C/T ACTTTAAGT r:TTTTTATTA C ACTTTAAGT Intron 4 AF136836.1#132 f:AAAATGTAT A TATGTATAA f:AAAATGTAT A/G TATGTATAA f:AAAATGTAT GTATGTATAA r:TTATACATA T ATACATTTT r:TTATACATA C/T ATACATTTT r:TTATACATAC ATACATTTT Intron 4 AF136836.1 #172 f:ATGATGTCT T ATTCATATT f:ATGATGTCTT/C ATTCATATT  f:ATGATGTCT C ATTCATATT r:AATATGAAT A AGACATCATr:AATATGAAT G/A AGACATCAT r:AATATGAAT G AGACATCAT Intron 4 AF136836.1#189 f:TTTATAGTT A TAATTTCAA f:TTTATAGTT A/G TAATTTCAA f:TTTATAGTT GTAATTTCAA r:TTGAAATTA T AACTATAAA r:TTGAAATTA C/T AACTATAAA r:TTGAAATTAC AACTATAAA Exon 5 AF136837.1 #42 f:CGAAGTTAC A TTAGGGAGA f:CGAAGTTACA/G TTAGGGAGA f:CGAAGTTAC G TTAGGGAGA r:TCTCCCTAA T GTAACTTCGr:TCTCCCTAA C/T GTAACTTCG r:TCTCCCTAA C GTAACTTCG Exon 5 AF136837.1 #101f:TCGGGACTT T ATCGATTGC f:TCGGGACTT T/G ATCGATTGC f:TCGGGACTT GATCGATTGC r:GCAATCGAT A AAGTCCCGA r:GCAATCGAT C/A AAGTCCCGA r:GCAATCGATC AAGTCCCGA Exon 5 AF136837.1 #104 f:GGACTTTAT C GATTGCTTC f:GGACTTTATC/G GATTGCTTC f:GGACTTTAT G GATTGCTTC r:GAAGCAATC G ATAAAGTCCr:GAAGCAATC C/G ATAAAGTCC r:GAAGCAATC C ATAAAGTCC Exon 5 AF136837.1 #117f:TGCTTCCTG A TCAAAATGG f:TGCTTCCTG A/T TCAAAATGG f:TGCTTCCTG TTCAAAATGG r:CCATTTTGA T CAGGAAGCA r:CCATTTTGA A/T CAGGAAGCA r:CCATTTTGAA CAGGAAGCA Exon6 AF136838.1 #309 f:CACTTCTAG G AAAAGGACA f:CACTTCTAGG/T AAAAGGACA f:CACTTCTAG T AAAAGGACA r:TGTCCTTTT C CTAGTTGTGr:TGTCCTTTT C/A CTAGTTGTG r:TGTCCTTTT A CTAGTTGTG Exon 7 NM_000770.1#1135 f:GTCCCCACC G GTGTGCCCC f:GTCCCCACC G/A GTGTGCCCC f:GTCCCCACC AGTGTGCCCC r:GGGGCACAC C GGTGGGGAC r:GGGGCACAC T/C GGTGGGGAC r:GGGGCACACT GGTOGGGAC Exon 7 AF136840.1 #232 f:AGGATAGGA G CCACATGCC f:AGGATAGGAG/T CCACATGCC f:AGGATAGGA T CCACATGCC r:GGCATGTGG C TCCTATCCTr:GGCATGTGG A/C TCCTATCCT f:GGCATGTGG A TCCTATCCT Exon 8 AF136842.1 #206f:ATGATGACA A AGAATTTCC f:ATGATGACA A/G AGAATTTCC f:ATGATGACA GAGAATTTCC r:GGAAATTCT T TGTCATCAT r:GGAAATTCT C/T TGTCATCAT r:GGAAATTCTC TGTCATCAT Exon 8 AF136843.1 #30 f:TGACCCTGG C CACTTTCTA f:TGACCCTGGC/T CACTTTCTA f:TGACCCTGG T CACTTTCTA r:TAGAAAGTG G CCAGGGTCAr:TAGAAAGTG A/G CCAGGGTCA r:TAGAAAGTG A CCAGGGTCA Exon 8 AF136843.1 #87f:GCCTTTCTC A GCAGGTAAT f:GCCTTTCTC A/G GCAGGTAAT f:GCCTTTCTC GGCAGGTAAT r:ATTACCTGC T GAGAAAGGC r:ATTACCTGC C/T GAGAAAGGC r:ATTACCTGCC GAGAAAGGC Intron 8 AF136843.1 #167 f:TACATGGCA C CTCCTCTGG f:TACATGGCAC/A CTCCTCTGG f:TACATGGCA A CTCCTCTGG r:CCAGAGGAG G TGCCATGTAr:CCAGAGGAG T/G TGCCATGTA r:CCAGAGGAG T TGCCATGTA Intron 8 AF136843.1#197 f:TTGCTATTT G TCCATGATC f:TTGCTATTT G/A TCCATGATC f:TTGCTATTT ATCCATGATC r:GATCATGGA C AAATAGCAA r:GATCATGGA T/C AAATAGCAA r:GATCATGGAT AAATAGCAA Intron 8 AF136843.1 #212 f:GATCAAGAG C ACCACTCTT f:GATCAAGAGC/T ACCACTCTT f:GATCAAGAG T ACCACTCTT r:AAGAGTGGT G CTCTTGATCr:AAGAGTGGT A/T CTCTTGATC r:AAGAGTGGT A CTCTTGATC Intron 8 AF136843.1#221 f:CACCACTCT T AACACCCAT f:CACCACTCT T/C AACACCCAT f:CACCACTCT CAACACCCAT r:ATGGGTGTT A AGAGTGGTG r:ATGGGTGTT G/A AGAGTGGTG r:ATGGGTGTTG AGAGTGGTG Intron 8 AF136843.1 #255 f:AATACACCA T CATTATTGG f:AATACACCAT/C CATTATTGG f:AATACACCA C CATTATTGG r:CCAATAATG A TGGTGTATTr:CCAATAATG G/A TGGTGTATT r:CCAATAATG G TGGTGTATT Intron 8 AF136843.1#271 f:TGGGCCAGA T AGCGGGGCT f:TGGGCCAGA T/C AGCGGGGCT f:TGGGCCAGA CAGCGGGGCT r:AGCCCCGCT A TCTGGCCCA r:AGCCCCGCT G/A TCTGGCCCA r:AGCCCCGCTG TCTGGCCCA Intron 8 AF136844. 1 #118 f:TTATTTACT G CATATTCTGf:TTATTTACT G/A CATATTCTG  f:TTATTTACT A CATATTCTG r:CAGAATATG CAGTAAATAA r:CAGAATATG A/G AGTAAATAA r:CAGAATATG T AGTAAATAA 3 UTRAF136845.1 #44 f:TCTGGCTGC C GATCTGCTA f:TCTGGCTGC C/T GATCTGCTAf:TCTGGCTGC T GATCTGCTA r:TAGCAGATC G GCAGCCAGA r:TAGCAGATC A/GGCAGCCAGA r:TAGCAGATC A GCAGCCAGA

TABLE 3 Comparison of allelic frequencies (%) from the populationsanalyzed (calculation based on Hardy-Weinberg law). SNP (−: rel. to ATG)(GenBank Acc. No African- ref. to, s. text) Caucasian Japanese AmericanLocation (−1731)-(−1732) 1.3 n.d. 11.7 5′UTR −1627  n.d. n.d. 16.6 5′UTR−1478  n.d. n.d. 2 5′UTR −1325  n.d. n.d. 1.4 5′UTR −1221  n.d. 1.6 n.d.5′UTR −1214  4.3 n.d. n.d. 5′UTR −1207  13.5 n.d. 3.75 5′UTR −1159  n.d.n.d. 1.1 5′UTR −1152  3.05 n.d. n.d. 5′UTR −980  n.d. n.d. 1.2 5′UTR(−765)-(−767) n.d. n.d. n.d. 5′UTR (−640)-(−641) 10.35 n.d. 4.5 5′UTR−411  14 37.8 17.4 5′UTR −370  19.2 29.7 3.2 5′UTR −271  23.3 5.7 3.25′UTR −248  n.d. n.d. 3.2 5′UTR −151  n.d. n.d. 1.6 5′UTR −133  n.d.n.d. 2.2 5′UTR −86 n.d. n.d. 1.2 5′UTR 171 1.4 n.d. n.d. Intron 1 258n.d. n.d. 1.6 Intron 1  122* Intron 2 150 n.d. n.d. 11.6 Intron 2180-181 30.8 51.1 47.8 Intron 2 182 n.d. n.d. 2.3 Intron 2 270 12 n.d.4.5 Exon 3 334 n.d. n.d. 15.9 Exon 3 378 14.6 n.d. 4.5 Intron 3  87 6.11.3 3.2 Intron 3 162 1.2 n.d. n.d. Intron 3 163 24.2 2.6 25.8 Intron 3 243^(§) Exon 4  13 n.d. n.d. 1.6 Exon 4 180 24.4 1.2 26.4 Intron 4 11629.6 46.4 7.4 Intron 4 132 4.7 n.d. 7.4 Intron 4 172 24.6 51.2 24.1Intron 4 189 2.6 n.d. n.d. Intron 4  42 n.d. n.d. 1.9 Exon 5 101 n.d.1.1 n.d. Exon 5 104 5 n.d. 1.7 Exon 5 117 n.d. n.d. 14.2 Exon 5 1135 n.d. n.d. 1.2 Exon 7 206 11.4 n.d. 3.2 Exon 8  30 n.d. 7.3 n.d. Exon 8 87 n.d. n.d. 2.17 Exon 8 167 6.3 n.d. 5.6 Intron 8 197 23 51.2 41.1Intron 8 212 1.3 n.d. n.d. Intron 8 221 n.d. 3.7 n.d. Intron 8 255 n.d.n.d. 1.1 Intron 8 271 2.1 n.d. 1.7 Intron 8 118 26.4 n.d. 44.4 Intron 8 44 22.3 53 n.d. 3′UTR *has been detected in a sample of a pre-screen(Caucasian sample). ^(§)has been detected in a phenotyped sample(Caucasian sample). n.d. - not detect in the samples analyzed.

Table 4 Listing of all amino acid changes in the coding regions (SEQ IDNOS: 1-2, 376-377, 3-16, 378-379, 17-18, 380-381 and 19-24,respectively, in order of appearance)

TABLE 4 Listing of all amino acid changes in the coding regions Met GlyLys Arg Ser Ile Glu s001.txt Exon 3 Met Gly Lys Lys Ser Ile Glu s002.txtGlu Leu Arg Lys Thr Lys Ala s376.txt Exon 3/4 Glu Leu Arg Lys Pro ArgLeu s377.txt Arg Lys Thr Lys Ala Ser Pro s003.txt Exon 3/4 Arg Lys ThrLys Ala Ser Pro s004.txt Ile Cys Ser Val Val Phe Gln s005.txt Exon 3 IleCys Ser Ile Val Phe Gln s006.txt Ala Ile Leu Asn Ser Pro Trp s007.txtExon 4 Ala Ile Leu Ser Ser Pro Trp s008.txt Arg Ser Tyr Ile Arg Glu Lyss009.txt Exon 5 Arg Ser Tyr Val Arg Glu Lys s010.txt Pro Arg Asp Phe IleAsp Cys s011.txt Exon 5 Pro Arg Asp Leu Ile Asp Cys s012.txt Arg Asp PheIle Asp Cys Phe s013.txt Exon 5 Arg Asp Phe Met Asp Cys Phe s014.txt CysPhe Leu Ile Lys Met Glu s015.txt Exon 5 Cys Phe Leu Phe Lys Met Glus016.txt Met Glu Gln Glu Lys Asp Asn s378.txt Exon 6 Met Glu Gln STOPs379.txt Val Pro Thr Gly Val Pro His s017.txt Exon 7 Val Pro Thr Ser ValPro His s018.txt Gln Asp Arg Ser His Met Pro s380.txt Exon 7 Gln Asp ArgIle His Met Pro s381.txt His Asp Asp Lys Glu Phe Pro s019.txt Exon 8 HisAsp Asp Arg Glu Phe Pro s020.txt Phe Asp Pro Gly His Phe Leu s021.txtExon 8 Phe Asp Pro Gly His Phe Leu s022.txt Met Pro Phe Ser Ala Gly Lyss023.txt Exon 8 Met Pro Phe Ser Ala Gly Lys s024.txt

1. An in vitro method for determining whether a human subject is at riskfor increased metabolism of a CYP2C8 substrate comprising the step of:(a) detecting in a CYP2C8 polynucleotide in the genome of the humansubject the nucleotide at the position corresponding to position 1668 ofSEQ ID NO: 400, wherein the presence of at least one copy of a G at saidposition indicates that the human subject is at risk for said increasedmetabolism of the CYP2C8 substrate relative to metabolism of the CYP2C8substrate of a human subject whose genome has two copies of a T at saidposition.
 2. The method of claim 1 further comprising the step ofdetecting the presence of a polypeptide encoded by the CYP2C8polynucleotide of the human subject.
 3. The method of claim 1, whereinthe detecting is performed using at least one of PCR, ligase chainreaction, restriction digestion, direct sequencing, nucleic acidamplification techniques, hybridization techniques and immunoassays. 4.The method according to claim 1, wherein the CYP2C8 substrate ispaclitaxel.